Ghrelin ameliorates cognitive deficits and restores dopamine responses to external stimuli in the PFC via D1 receptor signaling in Mecp2 KO mice, a mouse model for Rett syndrome

doi:10.21203/rs.3.rs-4838873/v1

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Ghrelin ameliorates cognitive deficits and restores dopamine responses to external stimuli in the PFC via D1 receptor signaling in Mecp2 KO mice, a mouse model for Rett syndrome

https://doi.org/10.21203/rs.3.rs-4838873/v1

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Rett syndrome is an X-linked neurodevelopmental disorder characterized by cognitive impairments along with sensory and motor deficits. Ghrelin is known to improve cognitive function in various animal models with cognitive deficits. Optimum activation of dopamine D1 receptor signaling in the prefrontal cortex (PFC) plays a critical role in cognitive performance. In this study, we investigated the effects of ghrelin on cognitive function and D1 receptor-mediated dopamine neurotransmission in the PFC of Mecp2 knockout (KO) mice, a mouse model for Rett syndrome. In the modified novel object recognition test, cognitive function was impaired in Mecp2 KO mice, and ghrelin injection (8.6 µg/mouse, s.c.) improved the cognition of objects and investigatory behaviors. In in vivo microdialysis studies, external stimuli such as saline injection and novelty induced increases in dopamine levels in the PFC of wild-type mice, and the dopamine release was bidirectionally regulated by D1 receptors. In the PFC of Mecp2 KO mice, the dopamine responses to external stimuli were attenuated and the dopamine reuptake system was upregulated. Pharmacological analyses revealed that the ability of D1 receptor signaling to inhibit dopamine release would be upregulated and/or its ability to stimulate dopamine release would be downregulated in Mecp2 KO mice. Ghrelin injection restored dopamine responses to external stimuli by adjusting the altered function of D1 receptor signaling. These results suggest that the ability of ghrelin to restore dopamine neurotransmission via D1 receptor-mediated mechanisms likely contributes to its therapeutic effects on cognitive deficits in Mecp2 KO mice.

Biological sciences/Neuroscience/Molecular neuroscience

Health sciences/Pathogenesis

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked methyl-CpG-binding-protein-2 (MECP2) gene, which encodes the transcriptional modulator MeCP2. Cognitive impairment is one of hallmarks in RTT [1], which is characterized by normal initial development and subsequent social and motor deficits, cardiac and gastrointestinal problems, a distorted breathing pattern and severe cognitive regression [2]. Central dopaminergic system has been implicated in cognitive control [3, 4]. Therefore, altered dopaminergic system is likely involved in cognitive impairment in RTT patients and animal models. Indeed, in mouse models for RTT, dysregulation of dopamine neurotransmission in the mesolimbic [5] and nigrostriatal [6] pathways associated with reward and motor function, respectively, has been reported. However, function of dopamine neurotransmission in the mesocortical pathway, which is associated with cognitive function, is not fully investigated despite cognitive impairment in RTT.

Ghrelin, an orexigenic peptide hormone predominantly secreted from the stomach [7], plays a key role in the regulation of neural plasticity and cognition [8]. For instance, the improvement of cognitive function by ghrelin or ghrelin receptor (growth hormone secretagogue receptor; GHSR) agonists has been reported in various animal models with cognitive deficits including Alzheimer's disease [9–12], Parkinson’s disease [13], depression [14], traumatic brain injury [15] and scopolamine-induced deficit [16]. However, the beneficial effect of ghrelin on cognitive deficits in developmental disorders such as RTT is unknown. Ghrelin and GHSR agonists affect dopamine neurotransmission in various brain regions including mesocorticolimbic pathways [17, 18]. Moreover, ghrelin amplifies dopamine D1 receptor signaling in neurons that coexpress D1 receptors and GHSRs, and the coexpression of D1 receptors and GHSRs is observed in neurons of the cortex and ventral tegmental area (VTA) [19]. Since an optimum activation of dopamine D1 receptor signaling in the PFC is required to improve cognitive performance [4], we hypothesized that the ability of ghrelin to amplify dopamine D1 receptor signaling in the PFC would improve cognitive deficits in Mecp2 KO mice by optimizing dopamine neurotransmission.

To test this hypothesis, we first screened the behavioral phenotype in Mecp2 KO mice, a mouse model for RTT, and cognitive impairment was detected [1]. Ghrelin administration to Mecp2 KO mice partially improved cognitive function. To understand mechanisms by which ghrelin improved cognitive function in Mecp2 KO mice, dopamine neurotransmission, especially the function of D1 receptors, was investigated using in vivo microdialysis. In this study, we are able to demonstrate that ghrelin has ability to restore suppressed dopamine neurotransmission by adjusting altered D1 receptor function in the PFC of Mecp2 KO mice.

Animals

Mecp2^−/+ female mice (B6.129P2(C)-Mecp2^tm1.1Bird/J strain) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and mated with C57BL/6 male mice [20, 21]. Genotyping was performed by PCR analysis of genomic DNA according to the protocol as described previously [21]. DNA samples were extracted from the tails of newborn mice after digestion with proteinase K. Male Mecp2^−/y (Mecp2 KO) mice and male Mecp2^+/y (WT) mice were maintained in a 12/12 h light/dark cycle with access to standard mouse chow and water ad libitum. Although most patients with RTT are female [2], male Mecp2 KO mice were used in this study, because male model mice displayed a robust phenotype consistently as described by others [22]. For instance, female mice heterozygous for Mecp2 deletion (Mecp2^−/+) develop their clinical phenotypes more slowly than male Mecp2 KO mice, and the onset of symptoms is variable among individuals, including cardiac abnormalities: QT_c prolongation was absent at 4 months, but present at 10 month [23], or absent at 11 months in some of them [24]. Thus, these features make characterization of phenotype more challenging in female Mecp2^−/+ mice. Male Mecp2 KO mice used for microdialysis to assess dopamine transmission in this study were at 9–13 weeks of age, most of which exhibited phenotype, unless otherwise noted in the figures. All mice were handled in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health, and the specific protocols were approved by the Institutional Animal Care and Use Committee of Kurume University. All efforts were made to minimize the number of animals used.

Drugs

Rat ghrelin acylated at the serine 3 position with an octanoyl group (PEPTIDE INC, Osaka, Japan) was dissolved in saline, and ghrelin at 8.6 µg/mouse was injected (s.c.) 30 min before the test. The dose of ghrelin was chosen based on the previous reports showing the induction of central dopamine release and behavioral modulation [25–27]. Cocaine (Takeda, Osaka, Japan), R(+)-SCH-23390 hydrochloride and (±)-SKF81297 (Sigma-Aldrich, St. Louis, MO) were dissolved in Ringer’s solution for application by retrograde microdialysis.

Surgery and Brain Dialysis

Microdialysis probes [I-shaped cannulas: customized home-made probes using dialysis membrane (Hospal Industrie, Meyzieu, France)] were implanted in the PFC (exposed length 2.8 mm) of mice under xylazine (8 mg/kg, i.p.) and sevoflurane (2–5%) anesthesia with local application of 0.1% lidocaine. The coordinates of the implantation were A/P + 1.9 mm, L/M 0.3 mm from the bregma and V/D -2.8 mm from the dura to target the PFC (Supplementary Fig. S1) [28]. As many of Mecp2 KO mice exhibit serious symptoms including respiratory disturbance and low body weight, general anesthesia and surgery easily resulted in intra-operative death, which was not observed in WT mice. Therefore, Mecp2 KO mice survived longer than 9 weeks with relatively less serious symptoms were predominantly used for brain surgery to prevent intra-operative death. After surgery, the mice were housed individually in acrylic cages (30×30×40 cm) for recovery.

Microdialysis experiments were performed 2 days after implantation of the probe. An online approach for real-time quantification of dopamine was used [29, 30]. The flow rate of Ringer’s solution was 2.0 µl/min. The 20-min sample fractions collected through the dialysis probes were directly injected to high-performance liquid chromatography using a reverse-phase column (150×4.6 mm, Supelcosil LC18; Merck, Darmstadt, Germany) with electrochemical detection. An LC-20AD pump (Shimadzu Corporation, Kyoto, Japan) was used in conjunction with an electrochemical detector (potential of the first cell, + 180 mV; potential of the second cell, -180 mV) (ESA, Chelmsford, MA). The mobile phase was a mixture of 4.1 g/L sodium acetate adjusted to pH 4.1, 100 mg/L Na₂EDTA, 120 mg/L octanesulfonic acid and 10% methanol. The flow rate was 0.4 ml/min. The detection limit of the assay was approximately 0.9 fmol per sample (on-column). The composition of Ringer’s solution was (in mM) NaCl 140.0, KCl 4.0, CaCl₂ 1.2, and MgCl₂ 1.0. At the end of the experiments, the mice were given an overdose of sevoflurane, and the brains were fixed with 4% paraformaldehyde via intracardiac infusion. Coronal sections (50 µm) were cut, and dialysis probe placement was localized using the atlas of Paxinos and Franklin [28] (Supplementary Fig. S1). Misplacement of the microdialysis probes was not observed in any experimental animals used for the study. The representative trace of the microdialysis probe location for the PFC is shown in Supplementary Fig. S1.

Novelty stimulus

During dopamine recording with in vivo microdialysis, a mouse was introduced to the novel acrylic plastic cage (30 cm (w) × 30 cm (d) × 30 cm (h)) for 20 min as a novelty stimulus, and then returned to the habituated acrylic plastic cage.

Behavioral tests

All tests were performed in the morning and in the same room.

Preferable novel object recognition (NOR) test

The conventional NOR test was modified for detection of cognitive abilities related to fear and vacancy in addition to recognizing novel and familiar objects. In addition to the novel and familiar objects in the conventional NOR test, ‘a fearful object’ and ‘no object’ were included. In the test box (44 cm (w) × 44 cm (d) × 44 cm (h)), four white plates (9 cm (w) × 9 cm (d)) were set 6 cm from the walls in symmetry. On these plates, different objects were stacked: (1) a small box made by four green half-cut disposal combs (Criterion XT precast gel, 18 well comb, Bio Rad) as a familiar object, which was put in the home cage the day before experiments; (2) a similar size and form blue box without comb incisions as a novel object; (3) four poles made by three marbles lined up vertically with glue for each as a fearful object; (4) no object. To test recognition and interest for the novel object, each experimental mouse was placed in the test box to explore the objects for 5 min. Animals were placed in the experimental room at least 30 min prior to testing. Field area of the test box was cleaned with 0.035% benzalkonium chloride solution and dried between subjects to avoid olfactory cuing. Movement information was obtained using SMART Video Tracking System (Harvard Apparatus, Holliston, MA) including the amount of time spent on each white plate area as exploring each object.

Open field test

Mice were placed in a square arena (42 cm (w) × 42 cm (d) × 42 cm (h)) made of white Plexiglass for 10 min in the light phase. Animals were placed in the experimental room at least 30 min prior to testing. The test was initiated by placing a single mouse in the middle of the arena and letting it move freely for 10 min. The arena was cleaned with 0.035% benzalkonium chloride solution and dried between tests. Movement information was obtained using SMART Video Tracking System.

Assessment of nest building

The performance in a nest-material manipulation task was evaluated as previously described [31, 32]. Briefly, one piece of Alpha-Nest made of cotton fiber (2 inch × 2 inch) (LBS Biotechnology, RH6 0UW, UK) was provided to each home cage with 3 each of WT or Mecp2 KO mice at 7–8 weeks of age. After 24 h, 48 h, 96 h, 7 days and 10 days, quality of the nests was scored by an observer. Nest quality was measured using the following four-point qualitative scale: 0: nest material untouched; 1: nest material nearly untouched; 2: nest material scattered, no clear shape evident; 3: nest of intermediate quality; 4: nest round and well built [32].

Phenotypic Scoring

Animals were scored using the RTT phenotypic severity scoring system [33]. Mice were scored from 0–2 for 8 parameters including wheezing, tremor, akinesia, hind limb clasping, posture, apnea and general condition (hair and eyes). A score of 0 represents the absence of symptoms, 1 indicates the presence of symptoms, and 2 indicates that the symptoms are severe [34].

Statistical Analysis

The data are displayed as the mean ± SEM. For analyses of the microdialysis data, all values were expressed as a percentage of the basal values (100%) for each group, which was obtained as the average of three stable baseline samples for dopamine. The values obtained after injection of saline or ghrelin were compared with the basal values using mixed models with time as a covariate, and Bonferroni’s correction was applied for multiple comparisons using the SAS MIXED procedure (version 9.4, SAS Institute, Cary, NC). Repeated measures two-way ANOVA was used to compare the experimental groups (GraphPad Prism 8.4.3, GraphPad Software, San Diego, CA). The basal values of dopamine were analyzed by one-way ANOVA and t-test with Welch’s correction (GraphPad Prism 8.4.3), as shown in Supplementary Table S1-S7. Behavioral data were analyzed by Student’s t-test, one-way ANOVA and two-way ANOVA using SigmaPlot 14.5 (Systat Software, San Jose, CA). P < 0.05 was considered to be significant.

Ghrelin increases interaction time with objects in the preferable NOR test in Mecp2 KO mice.

We first asked whether ghrelin has therapeutic efficacy for the behavioral phenotype of Mecp2 KO mice. In the preferable NOR test, WT mice spent more time exploring the novel object compared to the familiar or fearful object, regardless of ghrelin injection (8.6 µg/mouse, s.c.) (Fig. 1A top, 1B). Mecp2 KO mice showed no preference between novel and familiar objects, while they exhibited lower interaction time with the fearful object compared to the familiar object (Fig. 1B). These results suggest that cognition such as recognition memory is impaired, but a part of cognitive function such as innate risk-avoidance is functioning in Mecp2 KO mice (Blomeley et al., 2018). Ghrelin injection increased the interaction time with three objects (novel, familiar and fearful objects) in Mecp2 KO mice, but did not increase the time spent at no object area (Fig. 1B, 1C). Ghrelin may increase the cognition of objects and investigatory behaviors in Mecp2 KO mice. The time spent at center area in the open field test, an indicator of anxiogenic state, was similar between WT and Mecp2 KO mice, and this parameter was not affected by ghrelin, even when injected daily for three days (Fig. 1E). Distance moved during the test session of the preferable NOR test and the open field test in Mecp2 KO mice was lower than that in WT mice, and the low locomotor activity was not affected by ghrelin in either genotype (Fig. 1D, 1F).

Effects of ghrelin on general conditions were examined using the repeated ghrelin injection protocol (Supplementary Fig. S2A), which mimics the ghrelin administration protocol for patients with RTT [35]. Ghrelin (8.6 µg/mouse, s.c.) injected daily for three days followed by injection once a week while mice were alive did not affect the survival probability of Mecp2 KO mice (Supplementary Fig. S2B). Body weight of Mecp2 KO mice did not increase regardless of the repeated ghrelin injections (Supplementary Fig. S2C). Symptom and tremor scores increased over time regardless of the repeated ghrelin injections (Supplementary Fig. S2D, S2E), although tremor was improved by ghrelin in the clinical study [35]. Impaired nesting ability in Mecp2 KO mice at 9 weeks of age was not affected by the repeated ghrelin injection for 2 weeks (Supplementary Fig. S2F).

In summary, ghrelin improved the impaired cognition of objects in Mecp2 KO mice, but did not affect other behavioral phenotypes or general conditions. It has been proposed that the alteration of dopaminergic system involving D1 receptor signaling in the PFC is associated with the cognitive impairment [36]. Therefore, we investigated whether the dopaminergic system is altered in the PFC of Mecp2 KO mice, and, if altered, ghrelin can correct the abnormality of dopaminergic system.

Low response of dopamine in the PFC to external stimuli in Mecp2 KO mice

The basal levels of dopamine and its metabolites (DOPAC and HVA) in the PFC were similar between WT and Mecp2 KO mice (Supplementary Fig. S3). Saline injection increased dopamine levels up to 177% of basal levels in the PFC of WT mice, but failed to increase them in the PFC of Mecp2 KO mice (Fig. 2A). Exposure to the novel plastic cage (novelty stimulus) also increased dopamine levels up to 221% of basal levels in the PFC of WT mice, but the effects of the novelty stimulus were not statistically significant in the PFC of Mecp2 KO mice (Fig. 2B). These results demonstrate that Mecp2 KO mice exhibit the low response of dopamine to external stimuli in the PFC.

Ability of D1 receptor signaling to regulate dopamine release in the PFC is altered in Mecp2 KO mice.

To investigate whether the low response of dopamine to external stimuli in Mecp2 KO mice is due to the alteration of D1 receptor signaling, the effects of D1 receptor activation or inhibition on dopamine levels were examined in the PFC of WT and Mecp2 KO mice. Activation of D1 receptors by infusing a D1 receptor agonist, SKF81297 (1 µM), into the PFC induced the small increase in dopamine levels at 20 min, but did not affect dopamine levels at later time points in WT mice (Fig. 3A). In Mecp2 KO mice, SKF81297 (1 µM) gradually decreased dopamine levels over time (Fig. 3A). At a higher concentration (10 µM), SKF81297 continuously increased dopamine levels in WT mice, but decreased them in Mecp2 KO mice (Fig. 3B).

Next, the effects of D1 receptor inhibition were examined. Infusion of a D1 receptor antagonist, SCH23390 (1 µM), into the PFC did not affect dopamine levels in WT mice, but increased them after 100 min of SCH23390 infusion in Mecp2 KO mice (Fig. 3C). At a higher concentration of SCH23390 (10 µM), SCH23390 slightly increased dopamine levels after 80 min of infusion in WT mice, but largely increased them up to 457% in Mecp2 KO mice (Fig. 3D). The results clearly demonstrate that D1 receptor signaling in WT mice is involved in bidirectional regulatory mechanisms of dopamine release: the stimulatory and inhibitory pathways. In Mecp2 KO mice, the function of D1 receptors in the PFC is altered in Mecp2 KO mice: the ability of D1 receptor signaling to inhibit dopamine release would be upregulated and/or the ability to stimulate dopamine release would be downregulated.

Function of the dopamine reuptake system in the PFC is upregulated in Mecp2 KO mice.

We also evaluated the dopamine reuptake system in the PFC of Mecp2 KO mice. As cocaine is known to inhibit dopamine reuptake in the PFC via inhibition of the dopamine transporter and norepinephrine transporter [37, 38], the increase in dopamine levels induced by cocaine infusion into the PFC was compared between WT and Mecp2 KO mice. Cocaine at 1 and 10 µM increased dopamine levels in a concentration-dependent manner in the PFC of WT and Mecp2 KO mice (Fig. 4A, 4B). In Mecp2 KO mice, the increases in dopamine levels induced by cocaine infusion were larger than those in WT mice at both concentrations. The results suggest that the function of the dopamine reuptake system in the PFC is upregulated in Mecp2 KO mice.

Ghrelin restores dopamine responses in the PFC of Mecp2 KO mice via modulation of D1 receptor signaling.

To evaluate the effects of ghrelin on the low response of dopamine to external stimuli in the PFC of Mecp2 KO mice, ghrelin was administered prior to the novelty stimulus. In WT mice, ghrelin injection (8.6 µg/mouse, s.c.) as well as saline injection induced the increase in dopamine levels in the PFC (Fig. 5A). Although the maximum increase in dopamine levels induced by ghrelin injection was similar to that induced by saline injection, the ghrelin-induced increase in dopamine levels was maintained for longer time and observed even at 140 min after ghrelin injection. The novelty stimulus applied after ghrelin injection further increased dopamine levels (~ 200% of basal levels) over ghrelin-stimulated levels (~ 140% of basal levels) (Fig. 5A). When the dopamine levels before the novelty stimulus (100 to 140 min) were set at 100%, the novelty stimulus-induced increases in dopamine levels were similar between the saline and ghrelin groups (~ 150%) (Supplementary Fig. S4A). In Mecp2 KO mice, ghrelin injection, but not saline injection, increased dopamine levels in the PFC to a maximum of 190% of basal levels (Fig. 5B). The maximum effect of ghrelin was similar to that in WT mice, but the ghrelin-induced increases in dopamine levels returned to basal levels at 140 min after ghrelin injection. Dopamine levels in the PFC did not increase in response to the novelty stimulus after saline injection, but increased up to 216% of basal levels after ghrelin injection in Mecp2 KO mice (Fig. 5B). The results demonstrate that ghrelin is able to restore the dopamine responses to external stimuli in the PFC of Mecp2 KO mice.

As the abilities of D1 receptor signaling to inhibit and/or stimulate dopamine release is altered in the PFC of Mecp2 KO mice (Fig. 3), we asked the question whether ghrelin is able to adjust the alteration of D1 receptor signaling in Mecp2 KO mice. To answer this question, the effects of ghrelin was examined after infusion of SCH23390 (0.1 µM) into the PFC, at which concentration SCH23390 did not affect dopamine levels in the PFC of WT and Mecp2 KO mice (Supplementary Fig. S4B). In WT mice, SCH23390 reduced the ghrelin-induced maximum increase in dopamine levels and abolished the sustained increases in dopamine levels. The dopamine levels before the novelty stimulus were similar between the saline and ghrelin/SCH23390 groups, and the increases in dopamine levels in response to the novelty stimulus in the ghrelin/SCH23390 group were similar to those in the saline group (Fig. 5A). In WT mice, ghrelin induces dopamine release via mechanisms partly involving D1 receptor signaling, but ghrelin or inhibition of D1 receptor signaling does not affect the dopamine response to the novelty stimulus. In Mecp2 KO mice, the dopamine responses to ghrelin injection and to the novelty stimulus after ghrelin injection were completely abolished by the infusion of SCH23390 (0.1 µM) (Fig. 5B), suggesting that ghrelin has ability to restore the dopamine responses to external stimuli by adjusting the altered function of D1 receptor signaling.

This study demonstrated that cognitive function was impaired in Mecp2 KO mice, and ghrelin improved the cognition of objects and investigatory behaviors. An investigation of the mechanism for cognitive deficits revealed that the dopamine responses to external stimuli were attenuated and the dopamine reuptake system was upregulated in the PFC of Mecp2 KO mice (Fig. 6A, 6B). Ghrelin restored the dopamine responses to external stimuli in the PFC of Mecp2 KO mice. As the ability of D1 receptor signaling to inhibit dopamine release would be upregulated and/or its ability to stimulate dopamine release would be downregulated in the PFC of Mecp2 KO mice, ghrelin likely adjusted the altered function of dopamine D1 receptor signaling (Fig. 6B, 6C). Thus, ghrelin has beneficial effects on cognitive deficits via dopaminergic mechanisms in Mecp2 KO mice. The findings of this study proposes that ghrelin and GHSR agonists that increase dopamine D1 receptor signaling are promising pharmacological approach to improve cognitive function in RTT.

Effect of ghrelin on cognitive deficits in Mecp2 KO mice

Cognitive impact of ghrelin and GHSR agonists has been reported in animal models with a diverse range of pathology of cognitive deficits [8, 39, 40]. Consistently, ghrelin increased cognition of objects and investigatory behavior in Mecp2 KO mice in the present study, although the impairment of recognition memory was not improved. It is possible that cognitive function in Mecp2 KO mice could be more severely impaired compared to Mecp2R168X mutant mice, another mouse model for RTT [41], and other animal models for various pathologies of diseases. In fact, more sufficient effects of ghrelin on recognition memory were observed in animal models for Alzheimer's disease [10], sepsis [42] and dwarf [43].

The ghrelin/GHSR signaling pathway could be downregulated in the animal models with cognitive deficits. Indeed, ghrelin KO mice exhibit impairment of recognition memory [10] and ghrelin-O-acyl transferase (GOAT) KO mice, lacking an enzyme that catalyzes acyl modification to generate active form of ghrelin (octanoyl-ghrelin), exhibit impaired memory function [44]. So far, there is little information available about plasma ghrelin levels in MecP2 KO mice [45]. In humans, plasma levels of ghrelin are high during infancy, decrease until puberty and then increase to adult levels [46, 47]. In RTT patients, plasma levels of ghrelin continue to decrease after 10 years and are kept low during adulthood [46, 48] and low levels of plasma ghrelin reflect clinical symptoms such as eating difficulties [49]. Low levels of plasma ghrelin has also been reported in patients with cognitive deterioration suffering from mild traumatic brain injury [50], Parkinson's disease [51] and type 2 diabetes mellitus [52]. These studies in humans suggest that low levels of plasma ghrelin may be causally related to cognitive deficits in RTT patients. Developmental changes in plasma ghrelin levels and correlation between plasma ghrelin levels and the severity of cognitive deficits in MecP2 KO mice should be determined in future studies.

Dopaminergic pathology in the PFC of Mecp2 KO mice

Dopamine in the PFC is known to respond to nociceptive stimuli such as saline injection [30]. The dopamine response to external stimuli such as saline injection and novel environment observed in the PFC of WT mice is attenuated in the PFC of Mecp2 KO mice, suggesting that cognition of nociceptive stimuli and novel environment is impaired in Mecp2 KO mice. In RTT patients, decreased pain sensitivity is reported by parents and carers in two-thirds of subjects [53], although distinct alterations of pain sensitivity between external pain as being reduced and internal (visceral) pain as being increased have been noticed [53, 54]. Studies in animal models demonstrated that MeCP2 plays important roles in the function of nociceptive pathways via peripheral (e.g., the expression of MeCP2 and its epigenetic roles in the dorsal root ganglion after spared nerve injury [55–57]) and central (e.g., the expression of MeCP2 leading to repression of histone dimethyltransferase G9a and expression of BDNF in central nucleus of the amygdala after persistent pain [58]) mechanisms, and that 50% reduction of MeCP2 levels in the brain of Mecp2^flox/y mice induces the deficits in the pain recognition system [59]. It is likely that deficits in the central nervous system including decreased pain sensitivity and impaired cognition of external stimuli in Mecp2 KO mice cause the reduced dopamine responses in the PFC, and subsequently the reduced dopamine responses further impair cognitive function.

An optimum level of dopamine and activation of dopamine D1 receptor signaling in the PFC plays a key role in cognitive performance [4]. Cognitive deficits in Mecp2 KO mice can be in part attributable to the reduced dopamine responses to external stimuli in the PFC. The present study demonstrated that D1 receptors bidirectionally regulate dopamine release in the PFC of WT mice. In the PFC of Mecp2 KO mice, the ability of D1 receptor signaling to inhibit dopamine release would be upregulated and/or its ability to stimulate dopamine release would be downregulated, suggesting the alterations of D1 receptor signaling are mechanisms for the suppressed dopamine system.

The regulatory mechanism for dopamine release via D1 receptor signaling in the PFC has not been clarified. D1 receptors reside on different populations of pyramidal neurons in the PFC that project to striatal and other subcortical targets [60, 61]. Pyramidal neurons bidirectionally regulate the release of dopamine from mesocortical (VTA-PFC) dopamine neurons: activation of the PFC (Glutamate (Glu)) -basolateral amygdala (BLA) (Glu) -ventral pallidum (VP) (GABA) -VTA circuits [62] would result in inhibition of VTA dopamine neurons, whereas activation of the PFC (Glu) -ventral subiculum (vSub) (Glu) -NAc (GABA) -VP (GABA) -VTA circuits would result in activation (disinhibition) of VTA dopamine neurons [62]. If the function of D1 receptors in pyramidal neurons that activate the PFC-BLA-VP-VTA circuit or the PFC-vSub-NAc-VP-VTA circuit is upregulated or downregulated, respectively, the dopamine response to external stimuli in the PFC would be attenuated.

In addition, the PFC (Glu) -lateral habenula (Glu/trace amine) -rostromedial tegmental nucleus (RMTg) (GABA) -VTA circuit either inhibits or activates VTA dopamine neurons depending on whether stress conditions are chronic or acute, respectively [63]. D1 receptors are also present on GABAergic interneurons, most prevalently colocalizing with parvalbumin (PV)-positive interneurons in the PFC, which is involved in D1 receptor-mediated inhibition of the working memory circuit [64]. In Mecp2 KO mice, the inhibitory connections and functions of PV-positive interneurons on pyramidal neurons in visual cortex are upregulated, leading to the silencing of cortical circuits involved in vision [34, 65]. Alteration of D1 receptor function in PV-positive interneurons may potentially affect the inhibitory gating of various PFC-VTA circuits that regulate dopamine release in the PFC. Thus, functional alteration of D1 receptors expressed in subpopulation of pyramidal neurons or PV-positive interneurons and the biphasic neurotransmission of the PFC-VTA circuits that regulate dopamine release in the PFC would contribute to the attenuation of dopamine responses.

Experiments with cocaine infusions revealed that the function of dopamine reuptake sites, one of indicators for neuronal activity [66], is upregulated in the PFC of MecP2 KO mice (Fig. 6B (3)). The upregulation of the dopamine reuptake system would contribute, at least in part, to the low responses of dopamine to external stimuli in Mecp2 KO mice. Interestingly, in contrast to our findings, a marginal decrease in dopamine transporter density in the striatum of MecP2 KO mice was reported using positron emission tomography (PET) [67]. The modulatory role of dopamine D1 receptors in the dopamine reuptake system is not known, and their role in Mecp2 KO mice needs to be clarified in future studies.

Effect of ghrelin on dopaminergic pathology in the PFC of Mecp2 KO mice

Ghrelin is known to enhance dopamine D1 receptor signaling in neurons where GHSR and D1 receptors are coexpressed and form heterodimers [19]. Consistently, ghrelin restored the dopamine responses to external stimuli in Mecp2 KO mice via D1 receptor-dependent mechanisms. As cortical neurons coexpress GHSR and D1 receptors [19], it is possible that ghrelin treatment induces the formation of GHSR/D1 receptor heterodimers, leading the enhancement of D1 receptor signaling. In Mecp2 KO mice, the ability of D1 receptor signaling to inhibit dopamine release would be upregulated and/or its ability to stimulate dopamine release would be downregulated. The assumption of heterodimer formation supports the role of ghrelin in adjusting downregulated D1 receptor function (Fig. 6C (1)) more than upregulated D1 receptor function (Fig. 6C (2)).

Based on the assumption, ghrelin would preferentially restore the ability of D1 receptor signaling to stimulate dopamine release in response to external stimuli, leading to ameliorate cognitive deficits in Mecp2 KO mice. Behavioral evaluation of ghrelin effects with D1 receptor blockade will be necessary to confirm the importance of adjusting D1 receptor dysfunction for cognitive improvement. However, we could not perform the behavioral analyses with SCH23390 infusion, because the tubing connected to the infusion probe might interfere with the assessment of behaviors due to the muscle weakness and low body weight in Mecp2 KO mice. To overcome this limitation, Mecp2 KO mice at younger age prior to the onset of symptoms could be used for future studies.

The ability of ghrelin to downregulate the availability and function of dopamine transporters in Mecp2 KO mice (Fig. 6C (3)) cannot be ruled out, as they are critical to maintain extracellular dopamine levels [68]. However, the effects of ghrelin on dopamine transporters are not known at present.

In conclusion, ghrelin adjusts the altered D1 receptor function and subsequently restored the attenuated dopamine responses to external stimuli in the PFC of Mecp2 KO mice. The enhanced dopamine neurotransmission explains the action of ghrelin in ameliorating cognitive deficits in Mecp2 KO mice (Fig. 6). Ghrelin could be a potential therapeutic drug for cognitive deficits in patients with RTT and possibly other neurological disorders with cognitive deficits. Future preclinical studies are needed to explore whether the therapeutic benefits of ghrelin can allow for safe and sufficient clinical use in these conditions.

Acknowledgements

This work was supported by the NPO Rett syndrome support organization grant to YKa (2018-02-03), JSPS Grants-in-Aid for Scientific Research (KAKENHI) Grant Numbers JP19K08332 (YKa), JP20K08198 (TT), JP18K09756 (HK) and JP19H03410 (AN).

Ethics declarations

Competing interests

The authors declare no competing interests.

Contributions

YKa, TT, YY, TM and AN conceived the present project. YKa, YO and AN designed the experiments. YKa and YKi performed data analysis. YKa and YO performed the animal experiments. TT, YKi, KY, HK, YY, TM helped with animal experiments. YKa and AN wrote the manuscript.

Data availability statement

The datasets used in the present study are available upon reasonable request.

Ethics approval statement

All mice were handled in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health, and the specific protocols were approved by the Institutional Animal Care and Use Committee of Kurume University. All efforts were made to minimize the number of animals used.

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The authors have declared there is NO conflict of interest to disclose

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Ghrelin ameliorates cognitive deficits and restores dopamine responses to external stimuli in the PFC via D1 receptor signaling in Mecp2 KO mice, a mouse model for Rett syndrome

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Introduction

Materials and methods

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Drugs

Surgery and Brain Dialysis

Novelty stimulus

Behavioral tests

Preferable novel object recognition (NOR) test

Open field test

Assessment of nest building

Phenotypic Scoring

Statistical Analysis

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