Published papers

© 2020, The Author(s). Whilst the brain is assumed to exert homeostatic functions to keep the cellular energy status constant under physiological conditions, this has not been experimentally proven. Here, we conducted in vivo optical recordings of intracellular concentration of adenosine 5’-triphosphate (ATP), the major cellular energy metabolite, using a genetically encoded sensor in the mouse brain. We demonstrate that intracellular ATP levels in cortical excitatory neurons fluctuate in a cortex-wide manner depending on the sleep-wake states, correlating with arousal. Interestingly, ATP levels profoundly decreased during rapid eye movement sleep, suggesting a negative energy balance in neurons despite a simultaneous increase in cerebral hemodynamics for energy supply. The reduction in intracellular ATP was also observed in response to local electrical stimulation for neuronal activation, whereas the hemodynamics were simultaneously enhanced. These observations indicate that cerebral energy metabolism may not always meet neuronal energy demands, consequently resulting in physiological fluctuations of intracellular ATP levels in neurons.

© 2019 the authors. Selective serotonin (5-HT) reuptake inhibitors (SSRIs) are first-line antidepressants but require several weeks to elicit their actions. Chronic SSRI treatment induces desensitization of 5-HT1A autoreceptors to enhance 5-HT neurotransmission. Mice (both sexes) with gene deletion of 5-HT1A autoreceptors in adult 5-HT neurons (1AcKO) were tested for response to SSRIs. Tamoxifen-induced recombination in adult 1AcKO mice specifically reduced 5-HT1A autoreceptor levels. The 1AcKO mice showed a loss of 5-HT1A autoreceptor-mediated hypothermia and electrophysiological responses, but no changes in anxiety-or depression-like behavior. Subchronic fluoxetine (FLX) treatment induced an unexpected anxiogenic effect in 1AcKO mice in the novelty suppressed feeding and elevated plus maze tests, as did escitalopram in the novelty suppressed feeding test. No effect was seen in wild-type (WT) mice. Subchronic FLX increased 5-HT metabolism in prefrontal cortex, hippocampus, and raphe of 1AcKO but not WT mice, suggesting hyperactivation of 5-HT release. To detect chronic cellular activation, FosB + cells were quantified. FosB + cells were reduced in entorhinal cortex and hippocampus (CA2/3) and increased in dorsal raphe 5-HT cells of 1AcKO mice, suggesting increased raphe activation. In WT but not 1AcKO mice, FLX reduced FosB + cells in the median raphe, hippocampus, entorhinal cortex, and median septum, which receive rich 5-HT projections. Thus, in the absence of 5-HT1A autoreceptors, SSRIs induce a paradoxical anxiogenic response. This may involve imbalance in activation of dorsal and median raphe to regulate septohippocampal or fimbria-fornix pathways. These results suggest that markedly reduced 5-HT1A autoreceptors may provide a marker for aberrant response to SSRI treatment.

© The Author(s) 2018. Extracellular ATP, which is released from damaged cells after ischemia, activates P2 receptors. P2Y1 receptors (P2Y1R) have received considerable attention, especially in astrocytes, because their activation plays a central role in the regulation of neuron-to-glia communication. However, the functions or even existence of P2Y1R in microglia remain unknown, despite the fact that many microglial P2 receptors are involved in several brain diseases. Herein, we demonstrate the presence and functional capability of microglial P2Y1R to provide neuroprotective effects following ischemic stress. Cerebral ischemia resulted in increased microglial P2Y1R expression. The number of injured hippocampal neurons was significantly higher in P2Y1 R knockout (KO) mice than wildtype mice after forebrain ischemia. Propidium iodide (PI) uptake, a marker for dying cells, was significantly higher in P2Y1R KO hippocampal slices compared with wildtype hippocampal slices at 48 h after 40-min oxygen–glucose deprivation (OGD). Furthermore, increased PI uptake following OGD was rescued by ectopic overexpression of P2Y1R in microglia. In summary, these data suggest that microglial P2Y1R mediate neuroprotective effects against ischemic stress and OGD insult.

Background and Purpose: While the bladder vasculature is considered as a target of PDE5 inhibitors to improve bladder storage dysfunctions, its characteristics are largely unknown. Thus, the functional and morphological properties of arteries/arterioles of the bladder focusing on the NO-mediated signal transmission were explored. Experimental Approach: Diameter changes in rat bladder arteries/arterioles were measured using a video-tracking system. Intercellular Ca2+ dynamics in pericytes or smooth muscle cells (SMCs) of suburothelial arterioles were visualised using transgenic mice expressing GCaMP6 under control of the NG2- or parvalbumin-promoter. The perivascular innervation was investigated using fluorescence immunohistochemistry. Key Results: In rat suburothelial arterioles and vesical arteries, tadalafil (100 nM) attenuated nerve-evoked sympathetic vasoconstrictions. In both vascular segments, tadalafil-induced inhibition of sympathetic vasoconstriction was prevented by N ω-propyl-l-arginine hydrochloride (l-NPA, 1 μM), an nNOS inhibitor or N ω-nitro-l-arginine (l-NA, 100 μM). Both vascular segments were densely innervated with nNOS-positive nitrergic nerves in close apposition to tyrosine hydroxylase-immunoreactive sympathetic nerves. In pericyte-covered pre-capillary arterioles of the mouse bladder where sympathetic nerves were absent, nerve stimulation evoked transient reductions in pericyte Ca2+ levels that were shortened by l-NPA and abolished by l-NA. In SMC-containing arterioles, tadalafil (10 nM) caused a l-NPA-sensitive suppression of sympathetic Ca2+ transients. In mice, nitrergic perivascular nerves were distributed in the arterioles and the pre-capillary arterioles. Conclusion and Implications: Both nitrergic nerve and nerve-evoked endothelial NO release appear to be involved in vasodilatory signal transmission in bladder vasculature. The NO-mediated signal transmission is a potential target for PDE5 inhibitor therapy in bladder dysfunctions.

© 2020 The Authors Medium spiny neurons (MSNs) of mice show opposing activities upon the initiation of a food-seeking lever press task. Ventromedial striatal (VMS)-MSNs are inhibited but ventrolateral striatal (VLS)-MSNs are activated; these activities mediate action selection and action initiation, respectively. To understand what input shapes the opposing MSN activities, here, we monitor cortical input activities at the cell population level and artificially reverse them. We demonstrate that the ventral hippocampus (vHP) and the insular cortex (IC) are major inputs to the VMS and VLS, both projections show silencing at the trial start time, and the vHP-VMS and IC-VLS pathways form functionally coupled input-output units during the task. Of note, the upstream IC silencing is converted to the downstream VLS-MSN activation. We find biased localization of striatal parvalbumin-expressing interneurons (PV INs) and verify PV IN-dependent feedforward architecture in the VLS. Our results reveal a distinct mode of cortico-striatal signal conveyance via feedforward disinhibition in behaving animals.

© 2019 the authors. The axonal conduction of action potentials affects the absolute time it takes to transmit nerve impulses as well as temporal summation at destination synapses. At the physiological level, oligodendrocyte depolarization facilitates axonal conduction along myelinated fibers in the hippocampus; however, the functional significance of this facilitation is largely unknown. In this study, we examined the physiology of the facilitation of axonal conduction by investigating the changes in synaptic responses at destination synapses using male and female mice in which channelrhodopsin-2 expression was restricted to oligodendrocytes. The subiculum, one of the projection areas of the examined axons at the alveus of the hippocampus, is divided into three regions (proximal, mid, and distal) and contains two types of principal neurons: regular firing and bursting pyramidal cells.Wefound a significant increase in excitatory synaptic responses following optogenetic oligodendrocyte depolarization in bursting neurons at two of the three regions, but not in regular firing neurons at any region. The long-term potentiation (LTP) induced by theta burst stimulation at the synapses showing a significant increase was also enhanced after oligodendrocyte depolarization. Conversely, the reduction of oligodendrocyte depolarization during theta burst stimulation, which was achieved by photostimulation of archaerhodopsin-T expressed selectively on oligodendrocytes, reduced the magnitude of LTP. These results show that oligodendrocyte depolarization contributes to the fine control of synaptic activity between the axons they myelinate and targets subicular cells in a region- and cell type-specific manner, and suggest that oligodendrocyte depolarization during conditioning of stimuli is involved in the induction of LTP.

Optogenetic activation of serotonergic neurons in the dorsal raphe nucleus (DRN) enhances patience when waiting for future rewards, and this effect is maximized by both high probability and high timing uncertainty of reward. Here, we explored which serotonin projection areas contribute to these effects using optogenetic axon terminal stimulation. We found that serotonin stimulation in the orbitofrontal cortex (OFC) is nearly as effective as that in the DRN for promoting waiting, while in the nucleus accumbens, it does not promote waiting. We also found that serotonin stimulation in the medial prefrontal cortex (mPFC) promotes waiting only when the timing of future rewards is uncertain. Our Bayesian decision model of waiting assumed that the OFC and mPFC calculate the posterior probability of reward delivery separately. These results suggest that serotonin in the mPFC affects evaluation of time committed, while serotonin in the OFC is responsible for overall valuation of delayed rewards.

© 2019, The Author(s), under exclusive licence to Springer Nature America, Inc. The ability to sustain goal-directed action is essential for success in many domains, but little is known about the corresponding neural substrates. Using fiber photometry to monitor population neural activity, we demonstrate that engagement in sustained food- or punishment-motivated behavior is associated with suppression of ventral but not dorsal hippocampal activity. Using optogenetic stimulation, we demonstrate that this suppression is required for goal-directed behavior, whereas optogenetic suppression of the ventral hippocampus (vHP) enhances the ability to sustain goal-directed behavior. Suppression of vHP during sustained goal-directed behavior was accompanied by increased activity in median but not dorsal raphe, implicating serotonergic signaling through Htr3a as a mechanism of vHP suppression during successful goal-directed behavior. Sustainment of goal-directed action may require suppression of vHP because of the structure’s well-documented role in behavioral inhibition.

© 2019 The Authors. Psychiatry and Clinical Neurosciences © 2019 Japanese Society of Psychiatry and Neurology Apathy is a pervasive clinical phenomenon that deserves more attention at the translational and pre-clinical levels. To study apathy-like behavior in mice, we relied on an operational definition of apathy: the quantitative reduction of voluntary, goal-directed behaviors. We recently found that the chronic loss of function of a specific cell type (striatal dopamine receptor type 2-expressing medium spiny neurons, D2-MSN) in a restricted region (the ventrolateral striatum, VLS) was sufficient to induce apathy-like behavior in a food-seeking operant task. We further showed that VLS D2-MSN are activated at the preparatory period, and that optogenetic inhibition of VLS D2-MSN during that period resulted in transient decreases in instrumental motivation, strengthening the hypothesis that VLS D2-MSN mediate apathy-like behavior in mice. Mice bearing VLS D2-MSN dysfunction can thus be regarded as an apathy model for future translational studies.

Seizures invite seizures. At the initial stage of epilepsy, seizures intensify with each episode; however, the mechanisms underlying this exacerbation remain to be solved. Astrocytes have a strong control over neuronal excitability and the mode of information processing. This control is accomplished by adjusting the levels of various ions in the extracellular space. The network of astrocytes connected via gap junctions allows a wider or more confined distribution of these ions depending on the open probability of the gap junctions. K1 clearance relies on the K1 uptake by astrocytes and the subsequent diffusion of K1 through the astrocyte network. When astrocytes become uncoupled, K1 clearance becomes hindered. Accumulation of extracellular K1 leads to hyperexcitability of neurons. Here, using acute hippocampal slices from mice, we uncovered that brief periods of epileptiform activity result in gap junction uncoupling. In slices that experienced short-term epileptiform activity, extracellular K1 transients in response to glutamate became prolonged. Na1 imaging with a fluorescent indicator indicated that intercellular diffusion of small cations in the astrocytic syncytium via gap junctions became rapidly restricted after epileptiform activity. Using a transgenic mouse with astrocyte-specific expression of a pH sensor (Lck-E2GFP), we confirmed that astrocytes react to epileptiform activity with intracellular alkalization. Application of Na1/HCO3-cotransporter blocker led to the suppression of intracellular alkalization of astrocytes and to the prevention of astrocyte uncoupling and hyperactivity intensification both in vitro and in vivo. Therefore, the inhibition of astrocyte alkalization could become a promising therapeutic strategy for countering epilepsy development.

Reduced expression of glutamate decarboxylase 67 (GAD67), encoded by the Gad1 gene, is a consistent finding in postmortem brains of patients with several psychiatric disorders, including schizophrenia, bipolar disorder and major depressive disorder. The dysfunction of GAD67 in the brain is implicated in the pathophysiology of these psychiatric disorders; however, the neurobiological consequences of GAD67 dysfunction in mature brains are not fully understood because the homozygous Gad1 knockout is lethal in newborn mice. We hypothesized that the tetracycline-controlled gene expression/suppression system could be applied to develop global GAD67 knockdown mice that would survive into adulthood. In addition, GAD67 knockdown mice would provide new insights into the neurobiological impact of GAD67 dysfunction. Here, we developed Gad1tTA/STOP−tetO biallelic knock-in mice using Gad1STOP−tetO and Gad1tTA knock-in mice, and compared them with Gad1+/+ mice. The expression level of GAD67 protein in brains of Gad1tTA/STOP−tetO mice treated with doxycycline (Dox) was decreased by approximately 90%. The GABA content was also decreased in the brains of Dox-treated Gad1tTA/STOP−tetO mice. In the open-field test, Dox-treated Gad1tTA/STOP−tetO mice exhibited hyper-locomotor activity and decreased duration spent in the center region. In addition, acoustic startle responses were impaired in Dox-treated Gad1tTA/STOP−tetO mice. These results suggest that global reduction in GAD67 elicits emotional abnormalities in mice. These GAD67 knockdown mice will be useful for elucidating the neurobiological mechanisms of emotional abnormalities, such as anxiety symptoms associated with psychiatric disorders.

© 2018, Springer-Verlag GmbH Germany, part of Springer Nature. Rationale: Impulsive choice has often been evaluated in rodents according to the proportion of choices for the delayed large magnitude reinforcer (%large choice) in a delay-discounting task (DDT). However, because %large choice is influenced by both sensitivity to reinforcer magnitude and sensitivity to delayed reinforcement (i.e., discounting rate), distinctively evaluating such discounting parameters represents a critical issue demanding methods to determine each parameter in rats. The serotonin (5-HT) system is well known to be involved in impulsive choice; nevertheless, only a few studies have distinguished discounting parameters and investigated how 5-HT modulators affect discounting rate. Objective: Here, we performed a discounting parameter analysis in mice and examined the effects of various 5-HT modulators on discounting rate. Methods: We set up DDTs with different delay schedules to determine which schedule could address delay-discounting rates in mice. We examined the effect of the following drugs on impulsive choice: a 5-HT reuptake inhibitor (paroxetine), a 5-HT1A receptor agonist (8-OH-DPAT), and two 5-HT3 receptor antagonists (granisetron and ondansetron). Results: Mice showed typical delay discounting at the shorter delay schedules (up to 4 s delay). The %large choice under shorter, but not longer, schedules followed an exponential function and allowed us to derive discounting rates. We selected a DDT with a 4-s delay schedule for further experiments. Granisetron and ondansetron, but not paroxetine or 8-OH-DPAT, decreased discounting rates without affecting sensitivity to reinforcer magnitude. Conclusion: We found that a method to calculate discounting rates in rats is also applicable to mouse models. We also provided evidence that 5-HT3 antagonism controls impulsive choice in mice.

© 2018 The Author(s). A three-dimensional single-cell-resolution mammalian brain atlas will accelerate systems-level identification and analysis of cellular circuits underlying various brain functions. However, its construction requires efficient subcellular-resolution imaging throughout the entire brain. To address this challenge, we developed a fluorescent-protein-compatible, whole-organ clearing and homogeneous expansion protocol based on an aqueous chemical solution (CUBIC-X). The expanded, well-cleared brain enabled us to construct a point-based mouse brain atlas with single-cell annotation (CUBIC-Atlas). CUBIC-Atlas reflects inhomogeneous whole-brain development, revealing a significant decrease in the cerebral visual and somatosensory cortical areas during postnatal development. Probabilistic activity mapping of pharmacologically stimulated Arc-dVenus reporter mouse brains onto CUBIC-Atlas revealed the existence of distinct functional structures in the hippocampal dentate gyrus. CUBIC-Atlas is shareable by an open-source web-based viewer, providing a new platform for whole-brain cell profiling.

© 2018 Wiley Periodicals, Inc. Alexander disease (AxD) is a rare neurodegenerative disorder caused by gain of function mutations in the glial fibrillary acidic protein (GFAP) gene. Accumulation of GFAP proteins and formation of Rosenthal fibers (RFs) in astrocytes are hallmarks of AxD. However, malfunction of astrocytes in the AxD brain is poorly understood. Here, we show aberrant Ca 2+ responses in astrocytes as playing a causative role in AxD. Transcriptome analysis of astrocytes from a model of AxD showed age-dependent upregulation of GFAP, several markers for neurotoxic reactive astrocytes, and downregulation of Ca 2+ homeostasis molecules. In situ AxD model astrocytes produced aberrant extra-large Ca 2+ signals “AxCa signals”, which increased with age, correlated with GFAP upregulation, and were dependent on stored Ca 2+ . Inhibition of AxCa signals by deletion of inositol 1,4,5-trisphosphate type 2 receptors (IP3R2) ameliorated AxD pathogenesis. Taken together, AxCa signals in the model astrocytes would contribute to AxD pathogenesis.

© 2018 The Authors Although psychotropic drugs act on neurons and glial cells, how glia respond, and whether glial responses are involved in therapeutic effects are poorly understood. Here, we show that fluoxetine (FLX), an anti-depressant, mediates its anti-depressive effect by increasing the gliotransmission of ATP. FLX increased ATP exocytosis via vesicular nucleotide transporter (VNUT). FLX-induced anti-depressive behavior was decreased in astrocyte-selective VNUT-knockout mice or when VNUT was deleted in mice, but it was increased when astrocyte-selective VNUT was overexpressed in mice. This suggests that VNUT-dependent astrocytic ATP exocytosis has a critical role in the therapeutic effect of FLX. Released ATP and its metabolite adenosine act on P2Y 11 and adenosine A2b receptors expressed by astrocytes, causing an increase in brain-derived neurotrophic factor in astrocytes. These findings suggest that in addition to neurons, FLX acts on astrocytes and mediates its therapeutic effects by increasing ATP gliotransmission.

© 2018 The Authors To understand brain circuits of cognitive behaviors under natural conditions, we developed techniques for imaging neuronal activities from large neuronal populations in the deep layer cortex of the naturally behaving common marmoset. Animals retrieved food pellets or climbed ladders as a miniature fluorescence microscope monitored hundreds of calcium indicator-expressing cortical neurons in the right primary motor cortex. This technique, which can be adapted to other brain regions, can deepen our understanding of brain circuits by facilitating longitudinal population analyses of neuronal representation associated with cognitive naturalistic behaviors and their pathophysiological processes. Kondo et al. demonstrate calcium imaging from neuronal populations using a miniature fluorescence microscope in naturally behaving non-human primates (NHPs), common marmosets. This technique marks an advance beyond methods that use fixed head positioning, which limits the study of complex, self-determined behaviors such as social interactions or fear and anxiety.

© 2018 International Society for Neurochemistry Multiple sclerosis (MS) is an autoimmune disease characterized by immune-mediated inflammation, which attacks the myelin sheath. MS pursues a relapsing and remitting course with varying intervals between symptoms. The main clinical pathological features include inflammation, myelin sheath destruction and plaque formation in the central nervous system (CNS). We previously reported that cystatin F (CysF) expression is induced in demyelinating lesions that are accompanied by active remyelination (referred to as shadow plaques) but is down-regulated in chronic demyelinated lesions (plaques) in the spinal cord of MS patients and in several murine models of demyelinating disease. CysF is a cathepsin protease inhibitor whose major target is cathepsin C (CatC), which is co-expressed in demyelinating regions in Plp 4e/− mice, a model of chronic demyelination. Here, we report the time course of CatC and CysF expression and describe the symptoms in a mouse experimental autoimmune encephalomyelitis (EAE) model using CatC knockdown (KD) and CatC over-expression (OE) mice. In myelin oligodendrocyte glycoprotein (MOG)-EAE, CatC positive cells were found to infiltrate the CNS at an early stage prior to any clinical signs, in comparison to WT mice. CysF expression was not observed at this early stage, but appeared later within shadow plaques. CatC expression was found in chronic demyelinated lesions but was not associated with CysF expression, and CatCKD EAE mouse showed delayed demyelination. Whereas, CatCOE in microglia significantly increased severity of demyelination in the MOG-EAE model. Thus, these results demonstrate that CatC plays a major role in MOG-EAE. (Figure presented.).

© 2018 The Author(s) We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues. Tainaka et al. describe a strategy for developing hydrophilic tissue-clearing reagents based on comprehensive chemical profiling. The strategic integration of optimal chemical cocktails provided a series of CUBIC protocols, which enable the 3D imaging of the mammalian body and large primate and human tissues.

© 2020 The Authors. Hippocampus published by Wiley Periodicals LLC A number of studies have reported the involvement of the ventral hippocampus (vHip) and the lateral septum (LS) in negative emotional responses. Besides these well-documented functions, they are also thought to control feeding behavior. In particular, optogenetic and pharmacogenetic interventions to LS-projecting vHip neurons have demonstrated that the vHip→LS neural circuit exerts an inhibition on feeding behavior. However, there have been no reports of vHip neuronal activity during feeding. Here, we focused on LS-projecting vCA1 neurons (vCA1→LS) and monitored their activity during feeding behaviors in mice. vCA1→LS neurons were retrogradely labeled with adeno-associated virus carrying a ratiometric Ca2+ indicator and measured compound Ca2+ dynamics by fiber photometry. We first examined vCA1→LS activity in random food-exploring behavior and found that vCA1→LS activation seemed to coincide with food intake; however, our ability to visually confirm this during freely moving behaviors was not sufficiently reliable. We next examined vCA1→LS activity in a goal-directed, food-seeking lever-press task which temporally divided the mouse state into preparatory, effort, and consummatory phases. We observed vCA1→LS activation in the postprandial period during the consummatory phase. Such timing- and pathway-specific activation was not observed from pan-vCA1 neurons. In contrast, reward omission eliminated this activity, indicating that vCA1→LS activation is contingent on the food reward. Sated mice pressed the lever significantly fewer times but still ate food; however, vCA1→LS neurons were not activated, suggesting that vCA1→LS neurons did not respond to habitual behavior. Combined, these results suggest that gastrointestinal interoception rather than food-intake motions or external sensations are likely to coincide with vCA1→LS activity. Accordingly, we propose that vCA1→LS neurons discriminate between matched or unmatched predictive bodily states in which incoming food will satisfy an appetite. We also demonstrate that vCA1→LS neurons are activated in aversive/anxious situations in an elevated plus maze and tail suspension test. Future behavioral tests utilizing anxious conflict and food intake may reconcile the multiple functions of vCA1→LS neurons.

© 2019 Elsevier Ltd Apathy is clinically defined as lack of motivation. Apathy is a frequent symptom in patients with Alzheimer's disease (AD). It is unclear whether amyloid β (Aβ)pathology is associated with apathy. To address this question, we employed the AppNL-G-F/NL-G-F mouse, an Aβ deposition-bearing mouse without neurofibrillary tangles and neuronal cell death throughout the lifespan and used a progressive-ratio (PR)task to monitor instrumental motivation between the ages of 16 and 39 weeks. In the PR task, the number of lever presses to receive one reward increases and the number of active lever presses in the final trial a mouse completes represents a break point, which is an index of motivation. During the observation period, AppNL-G-F/NL-G-F mice overall did not show impaired motivation. However, AppNL-G-F/NL-G-F mice showed a dispersion of the break point at 39 weeks of age within the group. Therefore, we examined the association between the degree of the break point and Aβ pathology; the number of cored amyloid plaques in the striatum was inversely correlated with the degree of motivation. Furthermore, we measured the dopamine transporter (DAT)levels in the subcortical tissues including the striatum using western blot analysis and showed that AppNL-G-F/NL-G-F mice have lower DAT levels than do C57BL/6J mice. Although we could not directly determine the effect of core amyloid plaques on the DAT, the results of this study suggest a pathway through which cored amyloid plaques damage the DAT and cause impaired motivation. These results will draw attention to cored amyloid plaques and will aid researchers searching for new strategies that are effective for the prevention and treatment of impaired motivation.