The deletion of M4 muscarinic receptors increases motor activity in females in the dark phase

Abstract Objectives M4 muscarinic receptors (MR) presumably play a role in motor coordination. Previous studies have shown different results depending on genetic background and number of backcrosses. However, no attention has been given to biorhythms. Material and Methods We therefore analyzed biorhythms under a light/dark cycle obtained telemetrically in intact animals (activity, body temperature) in M4 KO mice growth on the C57Bl6 background using ChronosFit software. Studying pure effects of gene knockout in daily rhythms is especially important knowledge for pharmacological/behavioral studies in which drugs are usually tested in the morning. Results We show that M4 KO mice motor activity does not differ substantially from wild‐type mice during light period while in the dark phase (mice active part of the day), the M4 KO mice reveal biorhythm changes in many parameters. Moreover, these differences are sex‐dependent and are evident in females only. Mesor, night–day difference, and night value were doubled or tripled when comparing female KO versus male KO. Our in vitro autoradiography demonstrates that M4 MR proportion represents 24% in the motor cortex (MOCx), 30% in the somatosensory cortex, 50% in the striatum, 69% in the thalamus, and 48% in the intergeniculate leaflet (IGL). The M4 MR densities were negligible in the subparaventricular zone, the posterior hypothalamic area, and in the suprachiasmatic nuclei. Conclusions We conclude that cholinergic signaling at M4 MR in brain structures such as striatum, MOCx, and probably with the important participation of IGL significantly control motor activity biorhythm. Animal activity differs in the light and dark phases, which should be taken into consideration when interpreting the results.

mice for each MR subtype were generated and have been intensively studied . However, often contradictory results have been reported, particularly in terms of the role of M 4 MR in the motor activity control. Moreover, the changes in motor activity have been usually demonstrated in a short stretch of time.
The initial knockout study (Gomeza et al., 1999) strongly indicated that M 4 knockout significantly increases the overall animal motor activity. The increased locomotion of M 4 KO mice has been attributed to the enhanced dopaminergic signaling at D 1 dopamine receptors. Nevertheless, other M 4 KO study in which backcrossing was carefully performed showed no M 4 effects on motor activity (Woolley, Carter, Gartlon, Watson, & Dawson, 2009). A recent study in which a relatively long (30 min) evaluation of motor activity was performed showed an increase in motor activity (Koshimizu, Leiter, & Miyakawa, 2012). The initial studies were performed on mixed 129SvEv/CF-1 background while Koshimizu et al. (2012) worked with animals made on a pure 129SvEv background.
Knockout studies were initially considered as an optimal method for detection of gene function (Bymaster, McKinzie, Felder, & Wess, 2003). However, the flanking allele effect was not sometimes considered as an important factor for behavior determination (Crusio, Goldowitz, Holmes, & Wolfer, 2009). It is also necessary to stress that mice are nocturnal animals (Roedel, Storch, Holsboer, & Ohl, 2006), and thus, experiments performed in their nonactive phase can be affected by this fact.
It is sometimes difficult to compare the types of motor activity that are followed in different studies (open-field locomotor activity in boxes or on plus mazes, circadian activity on running wheels, or in cages). In general, all these motor activities are directed by similar mechanisms, and thus, it could give us the picture of differences in motor activity between different groups of mice. It has been shown previously that different types of locomotor activity are affected by sex steroid hormones. There were found differences in open field (Blizard, Lippman, & Chen, 1975), circadian genes expression (Kuljis et al., 2013), open field, light-dark transition test, running wheel, and elevated plus maze (Morgan & Pfaff, 2001). Concerning the mechanisms, female sex steroid (estrogen) has been shown to increase locomotor activity (Ogawa, Chan, Gustafsson, Korach, & Pfaff, 2003) and in open field (Morgan & Pfaff, 2001). Thus, we expected differences between males and females.
The most prominent structure is, of course, the suprachiasmatic nucleus (SCN). Other structures have been also implicated in these effects. There are areas with near proximity to SCN, such as the subparaventricular zone (SPVZ), the dorsomedial nucleus, and the posterior hypothalamic area (PHA) and the tuberomammillary nucleus (Abrahamson & Moore, 2006;Kramer et al., 2001). The striatum, the thalamus, and the intergeniculate leaflet (IGL (Hughes & Piggins, 2012;Morin, 2013)) are also areas with locomotor biorhythmic effects. The SCN is innervated by cholinergic nerves (Hut & Van der Zee, 2011), but does not need to be necessarily intrinsically cholinergic (van den Pol & Tsujimoto, 1985). It receives cholinergic projections from basal forebrain and brain stem tegmentum (Bina, Rusak, & Semba, 1993). There are species differences in the presence of cholinergic neurons in the SCN in rat, hamster, and mouse (Hut & Van der Zee, 2011).
We, therefore, studied activity and body temperature biorhythm under a light/dark cycle in well-defined C57BL/6 mice and in their counterparts lacking M 4 MR using a telemetric system that allowed us to see the pure knockout effect without the influence of handling or other manipulation. In addition to that this model can also show the effect of knockout on clear genetic background (see flanking allele effect described above). Studying pure effects in biorhythms is especially important knowledge for pharmacological and/or behavioral studies in which drugs/treatment or tests are usually performed in the morning (i.e., in the nonactive phase in mice).
We tested the hypothesis that M 4 MR affect the animal activity without an effect on body temperature. The basis for this comes from previously published data about M 4 KO mice that elicit similar hypothermic response as wild types (Bymaster et al., 2001). Moreover, we hypothesized that this effect can be seen in the dark period only (active part of the day), and, thus, the biorhythm characteristics would be changed accordingly. This hypothesis is based on the fact that M 4 MR are considered as receptors able to inhibit acetylcholine release (Bymaster et al., 2003). Acetylcholine levels are higher in the active period (Hut & Van der Zee, 2011). Thus, the lack of inhibitory M 4 MR would increase acetylcholine levels and, thus, increase locomotion in dark period. Last, we hypothesized that this difference is sexually dependent, because it has been previously shown that locomotor activity is affected by sex steroid hormones (see above).
One of the important questions in motor coordination regulation is the role of brain areas previously identified as connected with biorhythm regulation. Thus, we have performed autoradiography experiments and we compared MR density in several brain

| Animals
The mice lacking the M 4 muscarinic receptor were generated in Wess' laboratory (Gomeza et al., 1999) (Ma, Miao, & Novotny, 1998) which made the female group homogenous in hormone levels. Moreover, no differences were seen in actograms in females during 15 consecutive days.

| Telemetry
In order to judge the biorhythm changes in intact animals, we employed a telemetric apparatus to measure body temperature and overall motor activity.

| Biorhythm analysis
The data collected by telemetry were grouped into 10-min sequences, and the calculated means were used for further analysis.
The analysis was performed using the ChronosFit program (Arraj & Lemmer, 2006) employing Fourier analysis and the stepwise regression technique. Then, the data were transferred into the GraphPad Prism 5.04 program (San Diego, USA) for further statistical analysis.

| Receptor autoradiography
For receptor determination, autoradiography was performed in sev- and stored in storage boxes at −80°C until use. For binding to MR, the sections were allowed to thaw and dry for 30 min at 22°C and the density of receptors was determined as previously described (Farar & Myslivecek, 2016;Farar et al., 2012;Valuskova, Farar, Forczek, Krizova, & Myslivecek, 2018). In brief, sections were incu- Measurements were taken and averaged from at least three sections for each animal and brain region.

| Histology
Nissl staining was used for SCN, SPVZ, IGL, and PHA identification in MR autoradiography determination. In brief, the parallel sections were obtained using cryostat (the appropriateness of section was controlled using mice atlas (Paxinos & Franklin, 2008)), the sections were collected and divided into four sets. The first section from the set was placed on the first glass slide and used for Nissl staining, The area, clearly visible as in Nissl staining, was then marked (using border transposition) on a scanned autoradiogram and used for densitometry with PC-based analytical software (MCID software).

| Statistical analysis
As some variables from biorhythm analysis revealed dependency as also verified by Pearson's r (close to unity), we have used one-way ANOVA for analysis with post hoc Sidak's corrections. Values of p < 0.05 were considered significant. If the variables were independent, then there was statistical significance between the groups determined using the Student t test (WT vs. KO animals). In the same way, p < 0.05 was considered significant.

| Males
The biorhythm in M 4 KO was changed only in a minor manner (as can be seen from Figure 1) in comparison with control animals (WT mice, see

| Females
In deep contrast to males (please compare curves shown in Figure 1 and data in Tables 1 and 2 Figure S1) and find differences between these animals and in the power of the 24-hr period (ANOVA: F 3,52 = 23.88, p < 0.0001).

| Females versus males
It can be seen from Figure 2 that there was a difference between female and male overall activity. This can be seen in WT animals (ANOVA: F 7,120 = 29.98, p < 0.0001, see Tables 1 and 3), but to a much higher extent in KO animals (ANOVA: F 7,104 = 44.02, p < 0.0001, see Tables 1 and 4). There were common differences: in mesor (which was 1.42 times higher in WT females and 2.47 in KO females, respectively), nighttime mean (1.56 increase in WT females, 3.05 in KO females, respectively), and difference between night mean and day mean (N-D, 1.81 increase in WT females, 4.47 in KO females, respectively). In WT females (see Table 3  times) and amplitudes in 24-hr, 6-hr, 4.8-hr, and 4-hr rhythm.
Females also revealed higher power (ANOVA: F 3,52 = 23.88, p < 0.0001) of the 24-hr period when compared to males, as can be seen from periodograms shown in Figure 3.

| Males
With an aim to determine whether M 4 knockout specifically affects activity, we also followed the influence on body temperature.  Table S2) and with only minimal difference.

| Females
Similar to males, only a few parameters differed (see Supporting Information Table S3) between WT and KO females (Figure 4 left, bottom) although the extent of changes was higher than in males: maximal slope (KO had this value increased to 340% of control, t(37) = 3.182, p = 0.003) and 12-hr amplitude (KO had this value increased to 150% of control, t(30) = 2.274, p = 0.0303).

| Females versus males
It can be seen from Figure 4 (right above and bottom) that there was a slight increase in female compared to male temperature biorhythms. Although these increases were highly significant (ANOVA:

| MR density
KO females showed decrease ( Figure 5

| Histology
Representative sections comparing the histological and autoradiography picture are shown in Supporting Information Figure S2.

| D ISCUSS I ON
We demonstrate here that a lack of M 4 MR increases motor activity in the dark period and this effect is much more pronounced in females than in males. These biorhythm changes were specific as another biorhythm-temperature-did not differ between animals with deleted M 4 MR and control, that is, WT animals. Thus, there are no doubts about changed cholinergic signaling when M 4 MR are deleted as reported multiple times (Bymaster et al., 2001(Bymaster et al., , 2003Wess et al., 2003).
These findings are, to our knowledge, new.
We used entrained rhythms under a light/dark cycle, and, as we have noticed above, some biorhythm parameters reveal dependency, which was taken into account in our analysis. However, there are also some mutually interconnected parameters, which should be mentioned, like higher activity during the dark period that will result in higher power in the 24-hr period, which was found when comparing females to males.
Compelling evidence suggests an important role of the cholinergic system in the control of locomotor activity (Beeri et al., 1995;Martins-Silva et al., 2011;Miyakawa, Yamada, Duttaroy, & Wess, 2001;Shapovalova, Kamkina, & Mysovskii, 2005  Moreover, M 4 KO males backcrossed to C57BL/6NTac genetic background do not differ with controls in the amount and diurnal pattern of sleep, locomotor activity, and temperature (Turner, Hughes, & Toth, 2010). To assess locomotor activity in undisturbed mice over a prolonged period (Turner et al., 2010), as we did, we employed a telemetric system. Consistent with this report, we did not find (for 11 generations, founders were mixed 129SvEv/CF-1 background) to the C57BL/6NTac strain showed similar basal locomotor activity as their wild-type counterparts .
In general, locomotor activity affects body temperature to some degree. However, while the activity was changed at least by one half, the temperature was not changed or changed to a minor extent only (compare data in Tables 1, 3, 4, and Supporting Information Tables   S4 and S5). It is therefore probable that the M 4 MR effect is specific to activity but not to temperature that is directed by other MR (at least partly, i.e., in hypothermic response), by M 2 muscarinic subtype .
We have found a brain area-specific decrease in MR using non- and IGL in biorhythm regulation rather than in SCN, SPVZ, and PHA.
In an important way, IGL can provide feedback regulation (or fine tuning) of locomotor activity influencing SCN (Hughes & Piggins, 2012), and, thus, it should be stressed that its role in M 4 MR affected locomotor regulation.
Please note that although the density in TH is comparable to the density in SCN, we were able to detect about a one-half decrease in this area, indicating that we can detect a decrease even if the density in the area is quite low.
As muscarinic receptor subtype expression in the SCN is still a matter of debate, we clearly show here that the number of M 4 MR in the SCN is inappreciable. The initial paper that tried to detect MR in SCN used also autoradiography (Bina, Rusak, & Wilkinson, 1998).
These authors revealed that the muscarinic receptor density in the SCN is very low, mainly when compared to the striatum. We confirm this finding and are adding new knowledge about no M 4 MR presence in SCN. Another report indicated the presence of MR (generally) using immunohistochemistry (Hut & Van der Zee, 2011). It is not surprising that the PCR technique identified all five muscarinic receptor subtypes in the rat SCN (Yang, Wang, Cheng, Kuo, & Huang, 2010). The number of studies trying to identify the functional role of muscarinic receptor subtype in the SCN is limited. Carbachol, a muscarinic agonist, has been shown to induce phase advance in the circadian rhythm of spontaneous neuronal activity (Gillette et al., 2001), thus indicating the role of MR in the SCN. Taken together with our data, it should be another MR subtype that could be responsible for phase shift in the SCN, which also confirms with conclusion of Gillette et al. (2001), which suggested that this effect is Given the current understanding of M 4 modulation of dopamine signaling and evidence from M 4 KO mice, the impaired cholinergic control of dopamine signaling either directly in the striatum (Cachope & Cheer, 2014;Shin, Adrover, Wess, & Alvarez, 2015), or, in a more complex manner, involving polysynaptic circuits (Tzavara et al., 2004), can be suggested as the underlying mechanism affecting the motor activity and biorhythm in M 4 KO mice. M 4 MR are particularly abundant in striatum, where they modulate dopamine transmission.
The striatum is the main input structure of the basal ganglia circuitry network, processing inputs from several other brain areas including the whole cortical matter (Groenewegen, 2003 (Shin et al., 2015). In the striatum, M 4 serve as the main autoinhibitory receptors (Zhang et al., 2002).
We have hypothesized gender differences in motor coordination according to previous data (Kuljis et al., 2013). We have found only marginal changes in males, but clearly pronounced activity changes in females. There are also some other data showing gender differences in the running wheel, light-dark transition test, elevated plus maze, and open field (Blizard et al., 1975;Morgan & Pfaff, 2001;Ogawa et al., 2003). The effect on locomotor activity is mediated via the estrogen receptor α (Ogawa et al., 2003).
Moreover, morphological sex differences have been shown in the volume of the SCN (Gorski, Gordon, Shryne, & Southam, 1978). In an important way, gender differences in the 3 H-AFDX-384 binding sites have been found using autoradiography in striatum, nucleus accumbens, and olfactory tubercle (Fragkouli, Stamatakis, Zographos, Pachnis, & Stylianopoulou, 2006). More specific, although these authors (Fragkouli et al., 2006)   . Thus, we can consider the autoradiography binding in females as representative to brain areas responsible for locomotor regulation.
In an important way, the sex hormones have been shown to affect M 4 MR (El-Bakri et al., 2002). Ovariectomy upregulated M 4 MR in the hippocampal (dentate gyrus, CA1, CA3), hypothalamic structures, and in the frontal cortex. Estrogen substitution led to restoration of M 4 MR initial levels. In addition, ovariectomy decreased the exploratory (i.e., locomotor) activity of the rats that were restored by estrogen treatment. This can be hypothetically the reason for biorhythm changes in females: If ovariectomy upregulates M 4 MR and decreased activity, then M 4 MR knockout would have contrary effects. Progesterone treatment had no effect on the ovariectomy-induced upregulation of M 4 receptors.
Furthermore, some studies proved that circadian rhythmicity can be affected by sex hormones (Bailey & Silver, 2014), which are, per se, also the subject of rhythmicity. In the 80s, Wollnik (1985) observed obvious sex differences in the daily pattern of locomotion in laboratory rats. Hormonal and genetic differences between males and females also influence development of locomotor activity circadian rhythm (Diez-Noguera & Cambras, 1990). In the same way, estradiol has been shown to influence the level and distribution of daily locomotor activity, the response to light pulses behavior, and the time span of the free-running period (Blattner & Mahoney, 2014).
The nature of sex differences is not clear to date but hypothetically can also arise from higher androgen receptor (AR) expression in the SCN in males (Bailey & Silver, 2014).
Taking these data together with our results, we can conclude that non-SCN M 4 MR play a role in motor activity biorhythm regulation and that the IGL, together with the striatum and MOCx, is suspicious areas involved in this regulation.

This research was supported by Czech Science Foundation Grant
No. 17-03847S (to JM) and by PROGRES Q25/LF1/2 project by Charles University. We would like to thank Dr. Vladimir Riljak from our Institute for help with histology. Technical assistance by Katerina Janisova is greatly acknowledged.

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.