Abstract
Changes in the mother-offspring relationship are presumably accompanied by dynamic changes in the autonomic nervous system. Although temporal measurements of autonomic activity have been performed in human mothers and infants, the analysis of long-term changes remains unexplored. Mouse mothers can form social bonds with their pups and have a short period of pregnancy and lactation, which makes them useful for the examination of physiological changes from pregnancy to pup-rearing. Therefore, a telemetry system was used for several weeks to measure the changes in the autonomic nervous system and the behavior of mouse mothers. The current results showed that an electrocardiogram (ECG) could be stably recorded regardless of the movements of mothers and parturition. ECG analysis showed that the heart rate gradually decreased from pregnancy to lactation, and sympathetic activity sharply increased as the pups developed. Furthermore, the simultaneous recording of behavior and ECG in the home cage enabled us to understand the behavior-dependent influences on the ECG, thereby revealing the characteristics of autonomic nervous activity during each behavior. Thus, the present experimental method helps to understand how the physiological characteristics of mothers change from pregnancy through pup rearing, supporting the healthy development of pups.
Introduction
The mother-offspring relationship is unique among the relationships established by various animal species owing to its great impact on the future of the offspring1. In humans, the development and internal/external behaviors of children are influenced by parenting style as well as the extent of abuse and neglect2,3. Similarly, in rodents, the quality of maternal behavior has a significant impact on pup development and behavior4,5,6. Therefore, detailed tracking and examination of the nurturing behaviors of mothers can provide insights into the mechanisms of individual differences in the development and healthy support of their offspring.
Behavioral and physiological studies have shown that mammalian mothers undergo dynamic behavioral and physiological changes from pregnancy to lactation. When female mammals become pregnant, the secretion of estrogen and other hormones changes influence maternal behavior7. As the offspring grows and the frequency of lactation decreases, hormone secretion dynamically changes toward the pre-pregnant state, putting an end to the expression of maternal behavior8,9,10. These findings suggest that the interaction between the endocrine system, maternal behavior, and offspring development plays an important role in the changes that mammalian mothers experience during pregnancy and lactation.
Behavioral and physiological changes in mammalian mothers from pregnancy to lactation are closely related not only to the endocrine system but also to the autonomic nervous system11,12. Human studies suggest that mother-infant contact induces changes in the autonomic nervous system of both mothers and infants13. Several studies have measured the electrocardiogram (ECG) and heart rate variability in human mothers and infants, showing that each behavior alters the heart rate and RR interval of the others14,15,16. However, it is not clear how the three factors-autonomic nervous system, maternal behavior, and offspring development-interact with each other from pregnancy to lactation. Moreover, it is difficult to monitor these interactions in humans over a long period of time because the human lactation period is approximately two years.
Rodents are often used instead of humans in such studies. The autonomic nervous system of rodents has been measured under anesthesia or when isolated from pups to prevent unstable recording and damage to the measuring device; hence, the measurement is temporal under behaviorally restricted situations17,18,19. It is essential to observe the autonomic nervous system in an environment where rodents can move freely and communicate with others because mother-pup interactions can alter the behavior and physiology of mothers8,9,10,15.
This experimental method was developed to allow free movement of the mother. In this method, an ECG telemeter was attached subcutaneously to a pregnant mother to prevent damage to the device and allow stable long-term ECG recording from pregnancy to lactation. Mouse mothers can exhibit general behaviors (self-grooming, food intake, etc.) and usual maternal behavior in their home cage; hence, each behavior and ECGs can be observed and compared easily in the same mouse. A drive recorder recorded the mouse's behavior for 24 h over a period of four weeks. This experimental protocol allowed us to track the dynamic changes in autonomic activity and behavior from pregnancy to the mothering period.
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Protocol
All procedures were approved by the Ethics Committee of Azabu University (#210319-30). C57B/6J mice at gestational day (GD) 14 weighing over 22 g were used for the present study. The animals were obtained from a commercial source (see Table of Materials). The reagents and equipment needed for the study are listed in the Table of Materials.
1. Experimental preparation
- Turn on the panel heater and cover it with aluminum foil. Wipe all surfaces with 70% ethanol.
- Place all surgical instruments (scissors, forceps, fine tweezers, and tweezers) in a beaker containing 70% ethanol for sterilization.
- Disinfect the silk sutures (0.31 mm), which have been cut to lengths of 10 cm and 20 cm, by placing them in a 70% ethanol solution.
- Cover anesthesia masks with rubber masks to ensure sterility and patient comfort.
- Inspect the mouse biopotential telemeter (Figure 1A) for any abnormalities or damage using the tBase and LabChart (the physiological data analysis software) applications installed on a computer capable of recording.
2. Telemeter implantation
- Place the mouse in an induction anesthesia box with 4% isoflurane.
- After achieving anesthesia, place the mouse in a prone position on the panel heater covered with aluminum foil. Position the anesthesia mask on the mouse, maintaining an isoflurane level of 1%-2.0% and an oxygen flow at 0.5-2 L/min.
- Use tweezers to pluck the fur between the ears and disinfect the skin with 70% ethanol.
- Make an incision (2-3 cm) in the skin between the ears using scissors. Insert forceps into the incision to separate the skin and muscle around the neck and the ventral side, creating space on the ventral side specifically for telemetry placement.
- Insert the telemeter through the incision between the ears and place it in the ventral side space.
- Use forceps to roll and bundle the positive and negative leads. Place the leads in the neck space (Figure 1B).
- Suture the incision using a 13 mm needle and a 20 cm suture, taking care to avoid damage to the leads.
- Place the mouse in a supine position and pluck the fur around the clavicle with tweezers. Disinfect the skin using 70% ethanol.
- Make a small cut around the clavicle skin with scissors and separate the muscles from the neck skin. Extract the positive and negative leads from the back of the neck using forceps.
- Use forceps to separate the salivary glands and expose the "V-shaped" appearance of the sternocleidomastoid muscle (SCM), which originates from the clavicle and travels obliquely across each side of the neck (Figure 1C,D).
- Adjust the length of the negative lead to reach the SCM near the clavicle. Carefully remove the lead tubing from the negative lead, ensuring that the coiled stainless-steel electrode inside is stretched using fine tweezers.
- Pass the stitching needle (7 mm) with a 10 cm suture through the V-shaped bend of the SCM to create a loop. Pass the stainless-steel electrode from the negative lead through this loop and position it under the SCM near the clavicle.
- Lightly tie the loop to avoid damaging the SCM and repeat three times to secure the stainless-steel electrode. Secure the negative lead further by positioning the second suture on the cranial side of the SCM and tying it around the tubing of the negative lead (Figure 1E).
- Perform sectioning around the xiphoid process and carefully separate the skin from the muscle from the xiphoid process to the clavicle.
- Extend the positive lead to reach the xiphoid process. Similar to the negative lead, remove the lead tubing from the positive lead, ensuring the coiled stainless-steel electrode inside is stretched using fine tweezers.
- Use an 18 G needle to create a tunnel under the muscle around the xiphoid process, then place the stainless-steel electrode in a needle and pass it through the muscle around the xiphoid process.
- After removing the needle, lightly suture the coiled stainless-steel electrode to the muscle around the xiphoid process using a stitching needle (7 mm) with a 10 cm suture (Figure 1D). To further secure the positive lead, position the second suture on the cranial side of the xiphoid process and tie it around the tubing of the positive lead.
- Close all incisions using a stitching needle (13 mm) with a 20 cm suture length (Figure 1E,F).
- After implantation, place the mouse in a clean cage and position the cage on the tBase, which acts as the receiver for mouse telemetry (Figure 1G). Turn on the recordable computer and use the installed data analysis software application to collect ECG data. Check that the ECG data display normal waveforms (Figure 2A,B).
NOTE: In case of abnormal waveforms or noise (as shown in Figure 2C), reattach the negative and positive leads under anesthesia. - During the two-day recovery period, ensure the mouse is treated appropriately by placing water gel and food at the bottom of the cage. Record only home cage behavior with a drive recorder during this recovery period.
3. Recording of ECGs and home cage behavior from pregnancy to weaning
- Turn on the red lights during the dark phase because the drive recorder cannot function without light. Position the drive recorder by the home cage to record behavior. Record ECG using the LabChart application on the recordable PC. Before collecting the ECG data, verify the sampling rate of ECG in LabChart.
NOTE: In this protocol, the drive recorder was chosen because it can be placed anywhere. Additionally, compared to a video camera, drive recorders can record at a wide angle and use less data storage capacity (for example, recording 24 h with a drive recorder uses about 70 GB). In this demonstration, the sampling rate of ECG in the data analysis software is set at 1 k/s. - After turning on the drive recorder, launch the LabChart on the computer to record behavior and ECG.
NOTE: In this demonstration, a macro was utilized to continuously record ECG and automatically save the files every 2 h. This repetitive process was executed throughout the experiment. Additionally, a 256 GB SD card was selected for recording purposes, allowing for approximately 24 h of data collection per day. - Check the mouse for parturition and body abnormalities and sample the data from the drive recorder and the recordable computer. The day the mouse gave birth to their pups is considered as the postnatal day (PD) 0. Measure the weight of the pups every day after birth.
NOTE: In this study, the recording video from the drive recorder and ECG data file from the recordable PC were collected every morning from 7:30 to 8:30. Additionally, to prevent ECG noise from vibrations caused by multiple pups touching the mother, the litter was culled to four pups (half of them were male and the other half were female). - After checking the mouse and collecting the data, return everything to its original position. Turn on the drive recorder and the recordable computer. Start the LabChart application.
NOTE: If a macro is used to record ECG, click on the macro button and initialize the macro by selecting the run button from the manage option. - Once a week, replace the cage with a new one. During this process, include the bedding, half of which is previously used, and the other half is new.
NOTE: This process, including data sampling and mouse checks, was repeated from GD 17 to postnatal day (PD) 21.
4. Analyzing the ECGs
- To analyze the ECG data, use the the data analysis software. Start LabChart and open the data file for the recording period. To analyze heart rate variability (HRV), click on the HRV button and adjust the beat detection settings.
NOTE: In this demonstration, select the custom setting and change the minimum peak height to 1.2 for detection adjustment. - After adjusting the settings, review all beats and delete any noise data (as shown in Figure 2C).
NOTE: If an R wave is undetected, add or modify it using the HRV button. - Click on the HRV button and select the beat classifier view. Choose all beats on the beat classifier view, then select the report view from the HRV button. Copy the data from the report view and paste it into Excel.
NOTE: HRV results, as shown in Table 1, display both time and spectrum domains through the the data analysis software, enabling observation.
5. Categorizing the ethogram of home cage behavior
- Review all recorded videos to check for abnormalities, such as the red light not turning on during the dark phase and the drive recorder not working.
NOTE: The video is recorded in the same manner as the ECGs, with video clips captured every 2 h. - Categorize the ethogram based on parameters such as the mother's posture during behavior, time spent on behavior, and location where the behavior occurred (Table 2), and then observe the video from the drive recorder.
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Representative Results
After implanting the telemeter into the pregnant mouse, we recorded the ECGs from pregnancy to lactation in a home cage. The sampling rate was set to 1 k/s. To compare the ECG of each physiological state of the mother mouse while avoiding the influence of circadian rhythm, the 10 min data from 23:32 to 23:42 on GD 17, parturition, PD 0, and PD 21 from the 2 h data file (Figure 3, Table 1) were analyzed. The time from 23:32 to 23:42 was chosen as it represents the 10 min before the birth of the first pup during parturition. This specific time period was selected to demonstrate the feasibility of performing data analysis during parturition when muscle contractions were present. During all analysis phases, the mouse exhibited both general behaviors (drinking, digging, food, self-grooming) and maternal behaviors (nest building, licking/grooming), as observed from 6 out of the 16 ethograms listed in Table 2.
The ECGs of the mother were recorded without interference at GD17 when the fetus was inside the body (Figure 3A) and during parturition when physical changes such as muscle contractions occurred (Figure 3B). Additionally, the ECGs were recorded stably from pup birth (PD 0) to PD 21 (Figure 3C,D) despite various nursing behaviors. ECG analysis showed that the heart rate decreased gradually while the RR interval increased gradually from GD 17 to PD 21 (Table 1). The HRV analysis showed that the LF/HF ratio increased sharply from PD 0 to PD 21 (Table 1).
The ethograms of maternal and general behaviors recorded along with the ECG were subdivided, as shown in Table 2. We also confirmed that ECGs could be measured without waveform disturbances when the mothers exhibited any behavior defined in Table 2, such as feeding (Figure 4A,C) and crouching (Figure 4B,D).
Figure 1: Telemetry implanted at gestation day 14 in mouse. (A) Two leads were connected to a telemeter. The black represents negative lead, and the white represents positive lead. (B) The negative and positive leads were bundled and placed in the neck. The telemeter was placed on the ventral side. (C) The lead was removed from the neck, and the SCM was exposed. (D) The telemetry was positioned on the ventral side. The SCM is represented in pink. The clavicle and xiphoid process are represented in gray. The black line indicates the negative lead. The orange line indicates the positive lead. The blue line represents the suture position. (E) The negative lead was connected to the SCM. The positive lead was connected around the xiphoid muscle. (F) All incisions (represented by the red circle) were closed using a 20 cm suture. (G) Video capturing setup using a drive recorder. Please click here to view a larger version of this figure.
Figure 2: ECG recorded using the data analysis software. (A) The ECG was recorded for 3 s. (B) ECG for each heartbeat. (C) Red dashed lines indicate areas where the ECG was not recorded. Please click here to view a larger version of this figure.
Figure 3: ECG during pregnancy, parturition, and lactation. The ECG results of the same mouse at the same time.(A) ECG at GD 17. (B) ECG during parturition. (C) ECG at PD 0. (D) ECG at PD 21. Please click here to view a larger version of this figure.
Figure 4: ECG recording during behavioral observation. ECGs were recorded during the ethogram construction of the same mouse at PD 6.(A) Image of the mother eating solid food. (B) Image of the mother crouching over her pups. (C) The ECG of the mother while eating food. (D) The ECG of the mother while crouching over their pups. Please click here to view a larger version of this figure.
Table 1: Heart rate variability analysis based on ECG from pregnancy to lactation. Heart rate variability (HRV) analysis was performed based on the ECGs data shown in Figure 3. All analyses were shown for the same mouse and at the same time (23:32-23:42). Average RR, the average of the beat intervals; Average Rate, the average rate of the beat; SDSD, the standard deviation of the difference between successive beat intervals; RMSSD, root mean square of successive RR interval differences; pRR50, the percentage of beat interval differences greater than a fix duration; Total, the power contained within the entire frequency range; VLF, very low frequency; LF, low frequency; HF, high frequency; SD1; standard deviation along the minor axis of the Poincare distribution; SD2, standard deviation along the major axis of the Poincare distribution. Please click here to download this Table.
Table 2: Defined ethograms. All ethograms were defined based on parameters such as posture, time spent, and the place they visited. Please click here to download this Table.
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Discussion
In this method, wherein the telemeter was implanted into pregnant mice, the ECG could be continuously tracked in the same mouse from pregnancy to lactation. The mouse exhibited ethograms that included movement, indicating wakefulness during all analysis periods (23:32-23:42) from GD 17 to PD 21. Additionally, the present results showed that the heart rate gradually decreased from pregnancy to lactation. This decrease is considered to be due to the normalization of the heart rate, as several studies have shown that heart rates increase in pregnant females due to increased blood flow11,12.
Furthermore, sympathetic activity increased from PD 0 to PD 21 in the same mouse. However, there were no changes in sympathetic or parasympathetic activity from GD 17 to PD 0. Similar to the sympathetic data during pregnancy, previous studies have not reported any changes in sympathetic and parasympathetic activity in rats from day 4 to day 18 of pregnancy compared to those in virgin rats20. In mice, maternal autonomic activity may be modulated by the development of pups. However, further studies are required to determine whether these autonomic activity characteristics are common among mouse mothers or are influenced by the wakefulness state.
Some previous studies on mice measured ECGs during pregnancy and lactation with limitations on movement and natural behavior because the mother was isolated from the pups or under anesthesia17,18,19. Contrarily, the method employed in this study allowed the mother to move freely during ECG recording because the telemetry and electrode lead connected to the muscle were implanted subcutaneously. In particular, the ECG was recorded when the mother was eating and turning her head upward, even though the negative leads were attached to the SCM.
Additionally, implanting the device in vivo allowed stable recording, even with contact with cohabiting pups or vibrations caused by the activities of the mother. For example, the ECG was measured even when the mother exhibited self-grooming (data not shown) and crouching over the pups in the home cage. Based on these results, this method is effective for tracking the ECGs of mothers who spend their daily lives with their pups without any restrictions on their behavior. However, ECGs were not recorded when the telemetry used in this study was at a certain distance from the tBase; for instance, when the mothers were moving towards the food holder or climbing the cage. Therefore, improvements in the home cage environment are necessary.
This method also permits the recording of ECGs while performing activities such as maternal and general behaviors. To simultaneously record behavior and ECG in the home cage for long-term periods, a drive recorder5 was used instead of a video camera. Additionally, to analyze the recorded behaviors in detail, the ethograms were subdivided based on parameters such as posture, time spent, and other conditions for both maternal and general behaviors. The ECGs of mothers for each behavior could be divided and compared on the same day. This comparison highlights how the autonomic activity of a mother differs between maternal and general behaviors depending on the pregnancy-to-lactation period.
In conclusion, this protocol tracks the interaction among the three factors: autonomic activity, mother's behavior, and pup development, and it can provide insight into the characteristics and mechanisms of changes that occur in mothers from pregnancy to weaning.
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Disclosures
The authors declare no conflict of interest.
Acknowledgments
This study was supported by JSPS KAKENHI (Grant Numbers JP 21H04981 and 30974521) and the Center for Diversity, Equity & Inclusion, Azabu University.
Materials
| Name | Company | Catalog Number | Comments |
| 24-h repeating timer | Panasonic | WH3311BP | |
| Anesthesia box | Natsume Seisakusho Co | KN-1010 | W110×D110×H110mm |
| Anesthesia mask | Natsume Seisakusho Co | KN-1019-1 | |
| Anesthesic machine | Natsume Seisakusho Co | KN-1071-E | |
| C57BL6/J mice | Clea Japan, Inc | Pregnancy mouse at 14 day | |
| Clip light | Yazawa corporation | CLX60X02WH | |
| Configurator System | Adinstuments | TR190 | |
| drive recorder | Transcend | TS-DP250A-32G | |
| Food holder | Clea Japan, Inc | CL-2802 | |
| Isoflurane | FujiFilM | 099-06571 | |
| LabChart Pro V8 | Adinstuments | MLU260/8 | |
| LabChart8 | Adinstuments | MLS060/8 | |
| Mouse Biopotential Telemeter | Adinstuments | MT10B | |
| Needle 18 G 1 1/2 | Terumo | NN-1838R | |
| Panel heater | SANKO | 4976285145407 | |
| PowerLab 4/26 | Adinstuments | PL2604 | |
| Recordable computer | Mouse computer | mouse K7-H | |
| red light bulb | ELPA | LDG1R-G-GWP254 | |
| Rubber mask | Natsume Seisakusho Co | KN-1019-M | |
| SD card (256GB) | Transcend | TS256GUSD350V | It can record approximately 24 h |
| Silk suture 0.31 mm | Natsume Seisakusho Co | DMS2101 | |
| Suture needle 13 mm | Natsume Seisakusho Co | C-24-540-NO.0 | |
| Suture needle 7 mm | Natsume Seisakusho Co | C-24-540-NO.0000 | |
| tBase | Adinstuments | MT110 |
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