Sleep Deprivation Differentially Affects Dopamine Receptor Subtypes in Mouse Striatum

Abstract

The effects of sleep deprivation on dopaminergic systems remain elusive, in part due to the lack of selective ligands for dopamine receptor subtypes. We examined D₁, D₂ and D₃ receptor density in the mouse brain after sleep deprivation by receptor autoradiography using [³H]SCH 23390 for D₁R, [³H]raclopride for D₂R, and [³H]WC-10 for D₃R (a novel D₃R-selective compound developed in our laboratory, not previously reported in mouse). Sleep-deprived mice showed a significant decrease in D₁R, no change in D₂R, and a significant increase in D₃R binding in striatum. This pattern of dopamine receptor changes was not seen in mice subjected to restraint stress, suggesting specificity to sleep. These data provide evidence that brain dopaminergic circuits are remodeled after sleep deprivation.

INTRODUCTION

Multiple neurotransmitters are implicated in the sleep-wake cycle. There is recent evidence for the role of dopamine in the regulation of sleep and wakefulness. Microdialysis studies in rat have found that dopamine levels in the prefrontal cortex and nucleus accumbens exhibit a diurnal rhythm [1]. Mice lacking the dopamine transporter gene show increased wakefulness, while mice lacking the dopamine D₂ receptor show significantly decreased wakefulness [2,3]. Medications used to maintain wakefulness increase dopaminergic activity by primarily targeting the dopamine transporter reuptake mechanism [4]. In addition, patients with Parkinsons Disease, who show neurodegeneration of the striatonigral dopaminergic system, experience sleep disturbances including excessive daytime sleepiness [5]. Taken together, these studies together suggest a probable role for dopamine in the maintenance of wakefulness.

The effects of sleep deprivation on dopamine activity have recently been studied in the human brain using PET radiotracers. Healthy human subjects underwent one night of sleep deprivation and were imaged with [¹¹C]raclopride, which targets primarily D₂ receptors with less binding to D₃ receptors [6]. [¹¹C]raclopride binding was significantly reduced in the striatum and thalamus. Reduced binding suggests greater endogenous dopamine binding to D₂/D₃ receptors after sleep deprivation, possibly as a countermeasure to promote wakefulness during sleep deprivation [7]. However, the specific contribution of D₃R to sleep deprivation remains to be determined. Until this point it has been difficult to examine D₃R due to lack of selective pharmacologic tools.

Recently, a novel D₃R selective radioligand was developed in our laboratory, [³H]WC-10, or [³H]4-(Dimethylamino)-N-[4-(4-(2-Methoxyphenyl)Piperazin-1-yl)Butyl]Benzamide, a N-phenyl piperazine analog which displays high affinity and selectivity for D₃R [8–10]. The first quantitative autoradiographic analysis of the binding of [³H]WC-10 was performed in rat and rhesus monkey brain confirming localization of D₃R to the striatum, among other brain regions [9].

The aim of this study is to characterize the effects of sleep deprivation upon D₁R, D₂R, and D₃R binding in mouse striatum. As a comparison to sleep deprivation, we also examined the effects of restraint stress upon D₁R, D₂R, and D₃R binding in mouse striatum. This is the first report of the use of the [³H]WC-10 to localize D₃R binding in mouse.

METHODS

Animals

All experimental procedures involving animals were performed in accordance with guidelines established by the Animal Studies Committee at Washington University in St. Louis. All experiments were performed in a 12 hour dark and 12 hour light controlled room (lights on at 7:00 AM and lights off at 7:00 PM). Animals had access to food and water ad lib throughout the experiments.

Sleep deprivation

C57BL6 female mice at 2 months of age (n = 5) were sleep-deprived for 72 hours using the platform-over-water method as previously described [11]. Briefly, mice were placed on platforms consisting of a closed PVC pipe measuring 3.4 cm in diameter which was affixed to the bottom of a regular sized shoebox cage, with water filling the cage to 1 cm below the surface of the PVC pipe. This method has been validated by us and other laboratories with EEG/EMG electrode recording to substantially reduce both REM and non-REM sleep [11]. Age-and sex-matched control mice (n = 4) remained isolated in their home cages during the 72 hours. The 72 hour time point was chosen based on previous methodology using the platform-over-water method to induce chronic sleep deprivation [12]. Mice were then sacrificed and their brains immediately removed and fresh-frozen on powdered dry ice. Brains were sectioned at 30 μm on a cryostat, thaw-mounted onto Fisher Superfrost Plus slides, with 10 sets taken from the rostral through caudal striatum. Slides were stored at −80 °C until processing for receptor autoradiography.

Restraint stress

Age-matched two month old C57BL6 mice were randomly assigned to restraint stress or control groups (n=6 per group). Mice were subjected to restraint stress in a 50-mL conical tube for 2 h daily for 7 days in total as previously described [13,14], with the time point chosen to represent chronic stress. Control mice remained in their home cages. On the 7th day, all mice were sacrificed. Brains were removed, snap-frozen on powdered dry ice, and sectioned using a cryostat as described above.

Precursor synthesis and radiolabeling

[³H]WC-10 was synthesized by American Radiolabeled Chemicals (St. Louis, MO) by alkylation of the des-methyl precursor with [³H]methyl iodide. The specific activity of the radioligand was 80 Ci/mmol. The detailed synthesis scheme for [³H]WC-10 has been previously described [10].

Quantitative receptor autoradiography

All sections were pre-incubated for 20 min in buffer (50 mM Tris buffer, pH 7.4, 25C, containing 120 mM NaCl, 5 mM KCl) to remove endogenous dopamine binding. Following incubation with the respective tracer or non-specific binding buffers, slides were then rinsed five times at 1 min intervals with ice-cold buffer. Slides were incubated in an open staining jar as previously described, with the free radioligand concentration loss was less than 5% after ligands bound to brain sections, as previously described [9,10].

D₁ receptor binding

D₁ receptors were labeled with [³H]SCH 23390 using the procedure for rat brain described by Savasta et al. [15] with minor modifications. Specifically, brain sections were incubated for 15 min in a 7.4 pH buffer solution containing 50 mM Tris–HCl, 120 mM NaCl, 5 mM KCl, and 1 mM MgCl2 at room temperature. Sections were then incubated for 60 min at room temperature in a similar buffer solution with the addition of 1.5 nM [³H]SCH 23390 (PerkinElmer) and 30 nM ketanserin tartrate (Tocris) to block 5-HT₂ receptors. Non-specific binding was determined from the slides in the presence of 1 μM (+)-butaclamol, as described previously [9,16].

D₂ receptor binding

D₂ receptors were labeled with [³H]raclopride using the previously described procedure for rat and monkey tissue [9]. Brain sections were incubated for 60 min in buffer solution at room temperature with the addition of 4 nM [³H]raclopride (PerkinElmer). Non-specific binding was determined from the slides in the presence of 1 μM s(−)-eticlopride, as before [9].

D₃ receptor binding

D₃ receptors were labeled with [³H]WC-10 using the previously described procedure for rat and monkey tissue [9]. Brain sections were incubated for 60 min in buffer solution at room temperature with the addition of 4 nM [³H]WC-10. Non-specific binding was determined from the slides in the presence of 1 μM s(−)-eticlopride, as before [9].

Quantification of total radioactivity

Slides were air-dried and made conductive by coating the free side with a copper foil tape. Slides were then placed in the gas chamber with a mixture of argon and triethylamine (Sigma-Aldrich, USA) of a gaseous detector system, the Beta Imager 2000Z Digital Beta Imaging System (Biospace, France). After the gas was well mixed and a homogenous state was reached, further exposure for 24 hours yielded high-quality images. A [³H]Microscale (American Radiolabeled Chemicals, St. Louis, MO) was counted simultaneously as a reference for total radioactivity quantitative analysis. Quantitative analysis was performed with the program Beta-Vision Plus (BioSpace, France) for the anatomical region of interest.

A total of 7 to 8 brain sections were chosen for each animal. Using known neuroanatomical markers, bilateral regions of interest were drawn freehand along the border of the entire striatum of serial brain sections from each individual mouse brain to define the representative binding density for the striatum (see Figure 1A for representations of the coronal sections chosen). Data were linearly fitted to a standard curve which was then used for calibration with a coefficient (R) greater than 0.99, thereby converting cpm/mm into nCi/mg tissue. Subsequently, the receptor-bound radioligand densities were calculated utilizing the specific activity of each radioligand as previously described [9]. The experimenter was blinded to all conditions during the analysis.

Figure 1.

Changes in D₁, D₂ and D₃ receptor binding in mouse striatum after sleep deprivation. (A) Schematic mouse brain sections showing the rostral to caudal extent of the striatum, the region of interest in which D₁R, D₂R and D₃R were quantified, across a total of 7 sections (modified from [25]). (B) D₁ receptor autoradiography on multiple brain sections through the striatum shown in pseudocolor image intensity. D₁R binding was significantly decreased after sleep deprivation compared to controls (* p<0.05, two-tailed t-test). (C) D₂ receptor autoradiography on multiple brain sections through the striatum shown in pseudocolor image intensity. D₂R binding did not significantly change after sleep deprivation (p>0.05, two-tailed t-test). (D) D₃ receptor autoradiography on multiple brain sections through the striatum shown in pseudocolor image intensity. D₃R binding significantly increased after sleep deprivation compared to controls (* p<0.05, two-tailed t-test). Non-specific binding was negligible in all three dopamine receptor binding assays.

str = striatum; SD = sleep deprivation; NSB = non-specific binding.

Scale bars = 1 mm. Pseudocolor scale = 0 to 100% relative intensity.

RESULTS

Sleep Deprivation

Quantitative receptor autoradiography showed that [³H]SCH 23390, [³H]raclopride, and [³H]WC-10 labeled respective D₁R, D₂R, and D₃R throughout the rostral to caudal regions of the mouse striatum (Figure 1A–D).

Compared to age-matched controls, sleep-deprived mice showed an approximate 15% significant decrease in D₁ receptor density in the striatum (87 +/− 3 percent change relative to controls, per fmol/mg tissue, mean value +/− SEM, p < 0.05, t-test) (Figure 1B). There was no significant change in D₂ receptor density in the striatum (105 +/− 4 percent change relative to controls, per fmol/mg tissue, mean value +/− SEM, p > 0.05, t-test) (Figure 1C). In contrast, there was nearly a 20% significant increase in D₃ receptor density in the striatum (119 +/− 6 percent change relative to controls, per fmol/mg tissue, mean value +/− SEM, p < 0.05, t-test) (Figure 1D).

Restraint stress

Compared to age-matched controls, mice that underwent restraint stress showed no significant difference in D₁ receptor density in the striatum (95 +/− 3 percent change relative to controls, per fmol/mg tissue, mean value +/− SEM, p > 0.05, t-test) (Figure 2A). In contrast, there was a significant increase of approximately 25% in D₂ receptor density in the striatum (126 +/− 9 percent change relative to controls, per fmol/mg tissue, mean value +/− SEM, p < 0.05, t-test) (Figure 2B). Lastly, there was no significant difference in D₃ receptor density in the striatum (111 +/− 5 percent change relative to controls, per fmol/mg tissue, mean value +/− SEM, p > 0.05, t-test) (Figure 2C).

Figure 2.

Changes in D₁, D₂ and D₃ receptor binding in mouse striatum after restraint stress. (A) D₁R binding was not significantly changed after restraint stress compared to controls (p>0.05, two-tailed t-test). (B) D₂R binding significantly increased after restraint stress compared to controls (* p<0.05, two-tailed t-test). (C) D₃R binding was not significantly changed after restraint stress compared to controls (p>0.05, two-tailed t-test).

It should be noted that this pattern of dopamine receptor changes markedly differs from that seen after sleep deprivation. This suggests that dopamine receptor changes may be specific to sleep deprivation and not merely a byproduct of stress.

DISCUSSION

Until this point, it has been a challenge to elucidate the specific role of D₃R due to lack of selective radioligands available for receptor autoradiography. Our data are the first to show the full complement of D₁R, D₂R, and D₃R changes that occur together in the same animal after chronic sleep deprivation. This is also the first report of the localization of [³H]WC-10, the selective D₃R radioligand, in mouse striatum.

We show that total sleep deprivation for 72 hours decreases D₁R and increases D₃R density in the striatum, but has no significant effect on D₂R binding. Only one previous report has examined dopamine receptor changes after sleep deprivation, and this focused on D₁ and D₂ receptor binding after 96 hours of selective REM-sleep deprivation in rat [17].

One could argue that sleep deprivation is inherently stressful, and stress has well-established effects on dopamine receptor dynamics. However, mice that were subjected to chronic restraint stress did not show the same pattern of dopamine receptor changes that were seen after sleep deprivation. In contrast, stressed mice showed no such changes in D₁R or D₃R densities, but rather, they showed a significant increase in D₂R binding. This finding in stressed mice is consistent with previously published reports showing that both restraint stress and social stress significantly increase D₂R binding, but do not change in D₁R binding in rats [18]. Chronic stress has been reported to enhance dopamine uptake in the striatum, along with alterations in the dopamine transporter (DAT) activity [19]. Changes in DAT and D₂R density may represent homeostatic scaling mechanisms for the modulation of dopaminergic activity following periods of stress.

Dopamine receptors are a family of seven-transmembrane domain, G-protein coupled receptors. D₁-like dopamine receptors (D₁R and D₅R) are positive regulators of cAMP levels [20]. D₁R are thought to be exclusively post-synaptic in location. In contrast, D₂-like dopamine receptors (D₂R, D₃R, and D₄R) inhibit adenylyl cyclase activity in general. D₂R and D₃R are found both pre- and post-synaptically [21]. D₁, D₂ and D₃ receptors are extensively co-expressed in the striatum. It has been reported that D₁ and D₃ receptors may heterodimerize in the striatum, despite having opposing second messenger signaling cascades [22]. In the current manuscript, we observed that D₁ and D₃ receptors changed in opposite directions after sleep deprivation, whereas D₂ receptors remained unchanged. This finding invites renewed efforts in elucidating the functional roles of the different dopamine receptor subtypes in the brain.

Volkow et al. recently reported decreased [¹¹C]raclopride binding in humans after a single night of total sleep deprivation [7]. It would be interesting to image these subjects again after 72 hours to see if receptor changes persist, as we found for D₁R and D₃R density in mice. Our study results are not inconsistent with their finding of decreased [¹¹C]raclopride binding in humans; [¹¹C]raclopride was not used as a quantitative measure of D₂R density, but rather as an indirect measure of endogenous dopamine release [7]. Total sleep deprivation is thought to increase dopamine release in the striatum, whether as a consequence of, or perhaps even to help promote, wakefulness. Indeed, other studies have found that human sleep deprivation increases fMRI activation in the nucleus accumbens and is associated with risky decision making, which would be consistent with increased dopamine release [23].

The functions of the dopamine D₃R subtype in the brain are still being elucidated. Recent evidence uncovered a link between D₃R and a mouse model of restless legs syndrome, a sleep disorder with a circadian dysregulation of sensory and motor gating which is treated with dopaminergic agonist medications [24]. Other potential functions for D₃R include motor learning, motivation/reward, cognition, and locomotor response [22]. Given our results, future studies should be performed to better elucidate the specific role of D₃R and sleep.

SHORT CONCLUSION

Taken together, our data show that sleep deprivation differentially affects D₁, D₂, and D₃ receptor density in the striatum in a pattern that appears to be specific to sleep deprivation as opposed to generalized stress. In particular, D₃R changes may point to a novel role for D₃R in sleep behavior. These data provide further evidence that brain dopaminergic circuits may undergo remodeling during sleep deprivation.