A simultaneous recording of autonomic activity and detailed maternal behavior of mother mice from pregnancy to lactation was achieved using a telemetry system. This method helps to understand the dynamics of the physiological and behavioral characteristics in mothers from pregnancy to weaning.
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.
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.
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
2. Telemeter implantation
3. Recording of ECGs and home cage behavior from pregnancy to weaning
4. Analyzing the ECGs
5. Categorizing the ethogram of home cage behavior
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.
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.
The authors have nothing to disclose.
This study was supported by JSPS KAKENHI (Grant Numbers JP 21H04981 and 30974521) and the Center for Diversity, Equity & Inclusion, Azabu University.
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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