Variability in the Drug Response of M4 Muscarinic Receptor Knockout Mice During Day and Night Time

Abstract

Mice are nocturnal animals. Surprisingly, the majority of physiological/pharmacological studies are performed in the morning, i.e., in the non-active phase of their diurnal cycle. We have shown recently that female (not male) mice lacking the M₄ muscarinic receptors (MR, M₄KO) did not differ substantially in locomotor activity from their wild-type counterparts (C57Bl/6Tac) during the inactive period. Increased locomotion has been shown in the active phase of their diurnal cycle. We compared the effects of scopolamine, oxotremorine, and cocaine on locomotor response, hypothermia and spontaneous behavior in the open field arena in the morning (9:00 AM) and in the evening (9:00 PM) in WT and in C57Bl/6NTac mice lacking the M₄ MR. Furthermore, we also studied morning vs. evening densities of muscarinic, GABA_A, D₁-like, D₂-like, NMDA and kainate receptors using autoradiography in the motor, somatosensory and visual cortex and in the striatum, thalamus, hippocampus, pons, and medulla oblongata. At 9:00 AM, scopolamine induced an increase in motor activity in WT and in M₄KO, yet no significant increase was observed at 9:00 PM. Oxotremorine induced hypothermic effects in both WT and M₄KO. Hypothermic effects were more evident in WT than in M₄KO. Hypothermia in both cases was more pronounced at 9:00 AM than at 9:00 PM. Cocaine increased motor activity when compared to saline. There was no difference in behavior in the open field between WT and M₄KO when tested at 9:00 AM; however, at 9:00 PM, activity of M₄KO was doubled in comparison to that of WT. Both WT and KO animals spent less time climbing in their active phase. Autoradiography revealed no significant morning vs. evening difference. Altogether, our results indicate the necessity of comparing morning vs. evening drug effects.

Keywords: M4 muscarinic receptor; biorhythm; cocaine; motor activity; open field; oxotremorine; scopolamine; temperature.

FIGURE 1

The effects of scopolamine (SCO) on locomotor activity in WT (M₄WT) and KO (M₄KO) mice at 9:00 AM (top) and 9:00 PM (bottom). Control mice were treated with physiological solution (saline). Ordinate: counts per hour expressed as % of activity in saline injected mice. ^#p < 0.05, ^###p < 0.001, difference from PM.

FIGURE 2

The effects of oxotremorine on the maximal drop in temperature in WT (green columns) and KO (red columns) at 9:00 AM and at 9:00 PM (top). Ordinate: t_min [°C] minimal temperature. The effects of oxotremorine on the area under the curve (AUC) in WT (green columns) and KO (red columns) mice at 9:00 AM and at 9:00 PM (bottom). ^∗p < 0.05, ^∗∗∗p < 0.001, difference from WT mice, ^###p < 0.001, difference from the same strain at 9:00 AM.

FIGURE 3

The effects of oxotremorine/saline on temperature 360 min after oxotremorine application. The temperature was also monitored 60 min before drug application. Ordinate: t [°C], temperature. Abscise: time [min]. Top: the effects at 9:00 AM (oxotremorine/saline application); bottom: the effects at 9:00 PM (oxotremorine/saline application). No changes in temperature were recorded after saline application.

FIGURE 4

The effects of cocaine on locomotor activity in WT and KO mice at 9:00 AM (left) and 9:00 PM (right). Control mice were treated with physiological solution (saline), see legend in the figure. Ordinate: counts per hour expressed as % of activity in saline-injected mice. ^∗∗∗p < 0.001, difference from saline-injected mice.

FIGURE 5

Exploration of the novel area in the open field. The differences in WT (M₄WT) and KO (M₄KO) mice at 9:00 AM (left) and 9:00 PM (right). ^∗∗∗p < 0.001, difference from WT mice; ^#p < 0.05, difference from mice tested at 9:00 AM.

FIGURE 6

The duration of climbing in the Laboras apparatus (more detailed open field test). The differences in WT (M₄WT) and KO (M₄KO) mice at 9:00 AM (left) and 9:00 PM (right). ^###p < 0.001, difference from mice tested at 9:00 AM.

FIGURE 7

Changes in receptor densities (muscarinic receptors, kainate receptors, NMDA receptors, GABA_A receptors) revealed by autoradiography. Only significant results are shown. Motor cortex (MOCx), somatosensory cortex (SSCx), visual cortex (VisCx), striatum (caudate-putamen, CPu), nucleus accumbens (NAc), thalamus (TH), hippocampus (Hipp) and its specific areas CA3 and dentate gyrus (DG), and olfactory tubercle (OT). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001, difference from WT mice; ^#p < 0.05, difference from mice tested at 9:00 AM. See legend for group explanation.