[Advanced Neurocircuit Mapping via Non-invasive Magnetic Resonance Imaging Techniques].

The brain comprises a complex network of anatomically distinct regions (each with specialized functions) that collaborate to support various cognitive processes. Therefore, it is important to understand the brain from the perspective of a complex network. Functional magnetic resonance imaging (fMRI) is increasingly being accepted for its ability to provide useful insights into brain function. Among the fMRI techniques available in clinical practice, resting-state fMRI (rsfMRI) represents the core method for mapping brain activity in the absence of specific tasks; studies have reported the usefulness of rsfMRI in the investigation of various human diseases. Functional brain networks, which consist of interconnected regions that show correlated activities, are typically depicted as functional connectivity (FC). FC analysis using rsfMRI data provides extensive information, revealing intrinsic resting-state networks and highlights deviations in network structure among patients with psychiatric disorders. Such network insights not only deepen our understanding of the brain but also facilitate assessment of network alterations associated with psychiatric and neurodegenerative diseases.

Chemogenetic activation of mammalian brain neurons expressing insect Ionotropic Receptors by systemic ligand precursor administration.

Chemogenetic approaches employing ligand-gated ion channels are advantageous regarding manipulation of target neuronal population functions independently of endogenous second messenger pathways. Among them, Ionotropic Receptor (IR)-mediated neuronal activation (IRNA) allows stimulation of mammalian neurons that heterologously express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the original protocol, phenylacetic acid, a ligand of the IR84a/IR8a complex, was locally injected into a brain region due to its low permeability of the blood-brain barrier. To circumvent this invasive injection, we sought to develop a strategy of peripheral administration with a precursor of phenylacetic acid, phenylacetic acid methyl ester, which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. This strategy was validated by electrophysiological, biochemical, brain-imaging, and behavioral analyses, demonstrating high utility of systemic IRNA technology in the remote activation of target neurons in the brain.

Study on the Extrapolability of Current Tumorgenicity Test With Mice by Comparing the Syngeneic or Allogeneic Mouse Transplantation Model.

The extrapolability of the current tumorigenicity test performed by transplanting human cell product into immunodeficient (NOG) mice was investigated. For this purpose, the susceptibility to form teratomas of NOG mice was assessed by transplanting undifferentiated human-induced pluripotent stem cells (hiPSCs) as positive control cells via the liver, striatum, or tail vein and evaluating the TPD50 value (dose required to form teratomas in half of the transplanted mice). This was then compared to the TPD50 of syngeneic or allogeneic mouse models. The TPD50 of C57/BL/6(B6)-iPSC or 129/Ola(129)-embryonic stem cell (ESC) transplanted into the liver of syngeneic mice was 4.08 × 105 and 4.64 × 104 cells, respectively, while the TPD50 of hiPSC administered into the liver of NOG mice was 4.64 × 104 cells. The TPD50 of B6-miPSC-synergic, 129-mESC-synergic, or 129-cell/B6 allogeneic transplantation into the striatum was 5.09 × 102, 1.0 × 104, and 3.73 × 104 cells, respectively, while that of hiPSC/NOG mice was 1.0 × 103 cells. The TPD50 for B6-miPSC or 129-mESC syngeneic tail vein infusion was 3.16 × 106 or 5.62 × 106 cells, respectively, while no incidence was observed from 1 × 107 B6-miPSCs in 129 mice or hiPSCs in NOG mice infusion study. Although the number of data sets was limited, these data indicate that the teratoma formation from transplanted undifferentiated hiPSCs via the liver or striatum in NOG mice is comparable to that in syngeneic or allogeneic mouse transplantation model, suggesting that the result of the current tumorigenicity test in NOG mice would provide useful information to infer the incidence of teratoma from residual undifferentiated hPSCs in hPSC-derived products after transplantation.

Bone ingrowth into a porous structure is achieved by preceding fibrogenesis and vascularization.

Porous structures are frequently used in surgical implants to strengthen the interlocking power produced by bone ingrowth. Therefore, we aimed to elucidate the mechanism underlying bone ingrowth into a porous structure accompanied by vascularization. A nonbioactive polyetheretherketone implant with a 3D-printed porous structure was prepared and implanted in a bone hole created in the tibias of rabbits. We observed bone ingrowth in the same individual specimens immediately and at 2, 4, 8, and 12 weeks post-implantation using in-vivo computed tomography (CT). Furthermore, a detailed evaluation with blood vessels of each specimen at 2, 4, and 12 weeks was performed with ex-vivo CT and histological specimen. Additional histological evaluation was performed using thin sections of an implant made with thermoplastic polyurethane having the same structure. As a result, the bone invasion began after four weeks, when the construction of fibrous tissue and the spread of new blood vessels within the voids matured. As the bone matured in the load-bearing area, new blood vessels outside the bone matrix regressed. This longitudinal evaluation study suggests that preceding fibrogenesis and vascularization may be key in developing bone ingrowth. STATEMENT OF SIGNIFICANCE: A porous structure is an essential structure for dental and orthopedic implants because it provides strong fixation through bone invasion. Although it was known that vascularization was involved in this, the details were not known. This in vivo study revealed that in order for bone ingrowth to begin, a preparatory period of approximately 4 weeks was required to establish blood flow inside and outside the implant. Furthermore, it was confirmed that by spreading the fibrous structure in advance, it has an advantageous effect on the migration of cells involved in the formation of bones and blood vessels. We pointed out that it is necessary to consider fibrogenesis and vascularization when creating future implants.

Balancing risk-return decisions by manipulating the mesofrontal circuits in primates.

Decision-making is always coupled with some level of risk, with more pathological forms of risk-taking decisions manifesting as gambling disorders. In macaque monkeys trained in a high risk-high return (HH) versus low risk-low return (LL) choice task, we found that the reversible pharmacological inactivation of ventral Brodmann area 6 (area 6V) impaired the risk dependency of decision-making. Selective optogenetic activation of the mesofrontal pathway from the ventral tegmental area (VTA) to the ventral aspect of 6V resulted in stronger preference for HH, whereas activation of the pathway from the VTA to the dorsal aspect of 6V led to LL preference. Finally, computational decoding captured the modulations of behavioral preference. Our results suggest that VTA inputs to area 6V determine the decision balance between HH and LL.

Reorganization of Corticospinal Projections after Prominent Recovery of Finger Dexterity from Partial Spinal Cord Injury in Macaque Monkeys.

We investigated morphologic changes in the corticospinal tract (CST) to understand the mechanism underlying recovery of hand function after lesion of the CST at the C4/C5 border in seven macaque monkeys. All monkeys exhibited prominent recovery of precision grip success ratio within a few months. The trajectories and terminals of CST from the contralesional (n = 4) and ipsilesional (n = 3) hand area of primary motor cortex (M1) were investigated at 5-29 months after the injury using an anterograde neural tracer, biotinylated dextran amine (BDA). Reorganization of the CST was assessed by counting the number of BDA-labeled axons and bouton-like swellings in the gray and white matters. Rostral to the lesion (at C3), the number of axon collaterals of the descending axons from both contralesional and ipsilesional M1 entering the ipsilesional and contralesional gray matter, respectively, were increased. Caudal to the lesion (at C8), axons originating from the contralesional M1, descending in the preserved gray matter around the lesion, and terminating in ipsilesional Laminae VI/VII and IX were observed. In addition, axons and terminals from the ipsilesional M1 increased in the ipsilesional Lamina IX after recrossing the midline, which were not observed in intact monkeys. Conversely, axons originating from the ipsilesional M1 and directed toward the contralesional Lamina VII decreased. These results suggest that multiple reorganizations of the corticospinal projections to spinal segments both rostral and caudal to the lesion originating from bilateral M1 underlie a prominent recovery in long-term after spinal cord injury.

Characteristic Evaluation of a 11C-Labeled Leucine Analog, l-α-[5-11C]methylleucine, as a Tracer for Brain Tumor Imaging by Positron Emission Tomography.

Amino acid transporters are upregulated in many cancer cells, and system L amino acid transporters (LAT1-4), in particular, LAT1, which preferentially transports large, neutral, and branched side-chain amino acids, are considered a primary target for cancer positron emission tomography (PET) tracer development. Recently, we developed a 11C-labeled leucine analog, l-α-[5-11C]methylleucine ([5-11C]MeLeu), via a continuous two-step reaction of Pd0-mediated 11C-methylation and microfluidic hydrogenation. In this study, we evaluated the characteristics of [5-11C]MeLeu and also compared the sensitivity to brain tumors and inflammation with l-[11C]methionine ([11C]Met) to determine its potential for brain tumor imaging. Competitive inhibition experiments, protein incorporation, and cytotoxicity experiments of [5-11C]MeLeu were performed in vitro. Further, metabolic analyses of [5-11C]MeLeu were performed using a thin-layer chromatogram. The accumulation of [5-11C]MeLeu in tumor and inflamed regions of the brain was compared with [11C]Met and 11C-labeled (S)-ketoprofen methyl ester by PET imaging, respectively. Transporter assay with various inhibitors revealed that [5-11C]MeLeu is mainly transported via system L amino acid transporters, especially LAT1, into A431 cells. The protein incorporation assay and metabolic assay in vivo demonstrated that [5-11C]MeLeu was neither used for protein synthesis nor metabolized. These results indicate that MeLeu is very stable in vivo. Furthermore, the treatment of A431 cells with various concentrations of MeLeu did not change their viability, even at high concentrations (∼10 mM). In brain tumors, the tumor-to-normal ratio of [5-11C]MeLeu was more elevated than that of [11C]Met. However, the accumulation levels of [5-11C]MeLeu were lower than those of [11C]Met (the standardized uptake value (SUV) of [5-11C]MeLeu and [11C]Met was 0.48 ± 0.08 and 0.63 ± 0.06, respectively). In brain inflammation, no significant accumulation of [5-11C]MeLeu was observed at the inflamed brain area. These data suggested that [5-11C]MeLeu was identified as a stable and safe agent for PET tracers and could help detect brain tumors, which overexpress the LAT1 transporter.

Protocol for making an animal model of "blindsight" in macaque monkeys.

Patients with damage to the primary visual cortex (V1) can respond correctly to visual stimuli in their lesion-affected visual field above the chance level, an ability named blindsight. Here, we present a protocol for making an animal model of blindsight in macaque monkeys. We describe the steps to perform pre-lesion training of monkeys on a visual task, followed by lesion surgery, post-lesion training, and evaluation of blindsight. This animal model can be used to investigate the source of visual awareness. For complete details on the use and execution of this protocol, please refer to Yoshida et al. (2008)1 and Takakuwa et al. (2021).2.

Lewy Body Disease Primate Model with α-Synuclein Propagation from the Olfactory Bulb.

BACKGROUND: Lewy body diseases (LBDs), which are pathologically defined as the presence of intraneuronal α-synuclein (α-Syn) inclusions called Lewy bodies, encompass Parkinson's disease, Parkinson's disease with dementia, and dementia with Lewy bodies. Autopsy studies have shown that the olfactory bulb (OB) is one of the regions where Lewy pathology develops and initiates its spread in the brain. OBJECTIVE: This study aims to clarify how Lewy pathology spreads from the OB and affects brain functions using nonhuman primates. METHODS: We inoculated α-Syn preformed fibrils into the unilateral OBs of common marmosets (Callithrix jacchus) and performed pathological analyses, manganese-enhanced magnetic resonance imaging, and 18 F-fluoro-2-deoxy-d-glucose positron emission tomography up to 6 months postinoculation. RESULTS: Severe α-Syn pathology was observed within the olfactory pathway and limbic system, while mild α-Syn pathology was seen in a wide range of brain regions, including the substantia nigra pars compacta, locus coeruleus, and even dorsal motor nucleus of the vagus nerve. The brain imaging analyses showed reduction in volume of the OB and progressive glucose hypometabolism in widespread brain regions, including the occipital lobe, and extended beyond the pathologically affected regions. CONCLUSIONS: We generated a novel nonhuman primate LBD model with α-Syn propagation from the OB. This model suggests that α-Syn propagation from the OB is related to OB atrophy and cerebral glucose hypometabolism in LBDs. © 2022 International Parkinson and Movement Disorder Society.

Synthesis and Preclinical Evaluation of 18F-Labeled Ketoprofen Methyl Esters for Cyclooxygenase-1 Imaging in Neuroinflammation.

Cyclooxygenase (COX) is a rate-limiting enzyme in the synthesis of proinflammatory prostanoids from arachidonic acid. In vivo imaging of COX by PET is a potentially powerful tool for assessing the inflammatory response to injury, infection, and disease. We previously reported on a promising PET probe for COX imaging, 11C-labeled ketoprofen methyl ester, which can detect COX-1 activation in models of neuroinflammation and neurodegenerative disorders. In the current study, we aimed to design a fluorine-substituted benzoyl group of ketoprofen (FKTP) and to evaluate its racemate and enantiomers (18F-labeled ketoprofen methyl ester, [18F]FKTP-Me) as PET proradiotracers, potential radiopharmaceuticals for in vivo PET study of COX-1. Methods: We performed nucleophilic aromatic 18F-fluorination to obtain the desired racemic radiolabeled probe, (RS)-[18F]FKTP-Me, at a radiochemical yield of 11%-13%. Subsequent high-performance liquid chromatography separation with a chiral column yielded the desired enantiomerically pure (R)- and (S)-[18F]FKTP-Me. We examined the in vivo properties of (RS)-, (R)-, and (S)-[18F]FKTP-Me in PET studies using rats in which hemispheric inflammation was induced by intrastriatally injecting a lipopolysaccharide. Results: Racemic (RS)-[18F]FKTP-Me and enantiomeric (R)- or (S)-[18F]FKTP-Me were synthesized with radiochemical and chemical purities of more than 99%. The metabolite analysis revealed that the racemic (RS)-[18F]FKTP-Me crossed the blood-brain barrier and entered the brain, where it was subsequently hydrolyzed to its pharmacologically active acid form. PET images revealed a high accumulation of (R)-, (S)-, and (RS)-[18F]FKTP in the inflamed regions in rat brain. Moreover, the accumulated radioactivity of (S)-[18F]FKTP-Me was higher than that of (RS)-[18F]FKTP-Me and (R)-[18F]FKTP-Me, which was correlated with the stereospecific inhibitory activity of FKTP against COX-1. Conclusion: From the results of this study, we conclude that racemic (RS)-[18F]FKTP-Me and its enantiomers could act as proradiotracers of neuroinflammation in rat brain by the association of their hydrolyzed acid forms with COX-1 in inflamed regions. In particular, (S)-[18F]FKTP-Me demonstrated suitable properties as a COX-1-specific probe in PET imaging of neuroinflammation.

Insertable inductively coupled volumetric coils for MR microscopy in a human 7T MR system.

PURPOSE: To demonstrate the capability of insertable inductively coupled volumetric coils for MR microscopy in a human 7T MR system. METHODS: Insertable inductively coupled volume coils with diameters of 26 and 64 mm (D26 and D64 coils) targeted for monkey and mouse brain specimen sizes were designed and fabricated. These coils were placed inside the imaging volume of a transmit/receive knee coil without wired connections to the main system. Signal-to-noise ratio (SNR) evaluations were conducted with and without the insertable coils, and the g-factor maps of parallel imaging (PI) were also calculated for the D64 coil. Brain specimens were imaged using 3D T2∗ -weighted images with spatial resolution of isotropic 50 and 160 µm using D26 and D64 coils, respectively. RESULTS: Relative average (SD) SNRs compared with knee coil alone were 12.54 (0.30) and 2.37 (0.05) at the center for the D26 and D64 coils, respectively. The mean g-factors of PI with the D64 coil for the factor of 2 were less than 1.1 in the left-right and anterior-posterior directions, and around 1.5 in the superior-inferior direction or when the PI factor of 3 was used. Acceleration in two directions showed lower g-factors but suffered from intrinsic low SNR. Representative T2∗ -weighted images of the specimen showed structural details. CONCLUSION: Inductively coupled small diameter coils insertable to the knee coil demonstrated high SNR and modest PI capability. The concept was successfully used to visualize fine structures of the brain specimen. The insertable coils are easy to handle and enable MR microscopy in a human whole-body 7T MRI system.

Synthesis of L-[5-11 C]Leucine and L-α-[5-11 C]Methylleucine via Pd0 -mediated 11 C-Methylation and Microfluidic Hydrogenation: Potentiality of Leucine PET Probes for Tumor Imaging.

The efficient synthesis of L-[5-11 C]leucine and L-α-[5-11 C]methylleucine has been investigated using a continuous two-step sequence of rapid reactions consisting of Pd0 -mediated 11 C-methylation and microfluidic hydrogenation. The synthesis of L-[5-11 C]leucine and L-α-[5-11 C]methylleucine was accomplished within 40 min with a decay-corrected radiochemical yield of 15-38 % based on [11 C]CH3 I, radiochemical purity of 95-99 %, and chemical purity of 95-99 %. The Pd impurities in the injectable solution measured using inductively coupled plasma mass spectrometry met the international criteria for human use. Positron emission tomography scanning after an intravenous injection of L-[5-11 C]leucine or L-α-[5-11 C]methyl leucine in A431 tumor-bearing mice was performed. As a result, L-α-[5-11 C]methylleucine was found to be a potentially useful probe for visualizing the tumor. Tissue distribution analysis showed that the accumulation value of L-α-[5-11 C]methylleucine in tumor tissue was high [12±3% injected dose/g tissue (%ID/g)].

Disrupting tumor onset and growth via selective cell tagging (SeCT) therapy.

This study presents the early framework of selective cell tagging (SeCT) therapy, which is the concept of preferentially labeling specific cells in vivo with chemical moieties that can elicit a therapeutic response. Using glycosylated artificial metalloenzyme (GArM)-based protein labeling, this study reports two separate functional strategies. In one approach, early tumor onset can be suppressed by tagging cancer cells in living mice with an integrin-blocking cyclic-Arg-Gly-Asp (cRGD) moiety, thereby disrupting cell adhesion onto the extracellular matrix. In another approach, tumor growth in mice can be reduced by tagging with a cytotoxic doxorubicin moiety. Subsequent cell death occurs following internalization and drug release. Overall, experiments have shown that mouse populations receiving the mixture of SeCT labeling reagents exhibited a significant delay/reduction in tumor onset and growth compared with controls. Highlighting its adaptability, this work represents a foundational step for further development of SeCT therapy and its potential therapeutic applications.

The posterior parietal cortex contributes to visuomotor processing for saccades in blindsight macaques.

Patients with damage to the primary visual cortex (V1) lose visual awareness, yet retain the ability to perform visuomotor tasks, which is called "blindsight." To understand the neural mechanisms underlying this residual visuomotor function, we studied a non-human primate model of blindsight with a unilateral lesion of V1 using various oculomotor tasks. Functional brain imaging by positron emission tomography showed a significant change after V1 lesion in saccade-related visuomotor activity in the intraparietal sulcus area in the ipsi- and contralesional posterior parietal cortex. Single unit recordings in the lateral bank of the intraparietal sulcus (lbIPS) showed visual responses to targets in the contralateral visual field on both hemispheres. Injection of muscimol into the ipsi- or contralesional lbIPSs significantly impaired saccades to targets in the V1 lesion-affected visual field, differently from previous reports in intact animals. These results indicate that the bilateral lbIPSs contribute to visuomotor function in blindsight.

Divergent Whole Brain Projections from the Ventral Midbrain in Macaques.

To understand the connectome of the axonal arborizations of dopaminergic midbrain neurons, we investigated the anterograde spread of highly sensitive viral tracers injected into the ventral tegmental area (VTA) and adjacent areas in 3 macaques. In 2 monkeys, injections were centered on the lateral VTA with some spread into the substantia nigra, while in one animal the injection targeted the medial VTA with partial spread into the ventro-medial thalamus. Double-labeling with antibodies against transduced fluorescent proteins (FPs) and tyrosine hydroxylase indicated that substantial portions of transduced midbrain neurons were dopaminergic. Interestingly, cortical terminals were found either homogeneously in molecular layer I, or more heterogeneously, sometimes forming patches, in the deeper laminae II-VI. In the animals with injections in lateral VTA, terminals were most dense in somatomotor cortex and the striatum. In contrast, when the medial VTA was transduced, dense terminals were found in dorsal prefrontal and temporal cortices, while projections to striatum were sparse. In all monkeys, orbitofrontal and occipito-parietal cortex received strong and weak innervation, respectively. Thus, the dopaminergic ventral midbrain sends heterogeneous projections throughout the brain. Furthermore, our results suggest the existence of subgroups in meso-dopaminergic neurons depending on their location in the primate ventral midbrain.

Contribution of the Pulvinar and Lateral Geniculate Nucleus to the Control of Visually Guided Saccades in Blindsight Monkeys.

After damage to the primary visual cortex (V1), conscious vision is impaired. However, some patients can respond to visual stimuli presented in their lesion-affected visual field using residual visual pathways bypassing V1. This phenomenon is called "blindsight." Many studies have tried to identify the brain regions responsible for blindsight, and the pulvinar and/or lateral geniculate nucleus (LGN) are suggested to play key roles as the thalamic relay of visual signals. However, there are critical problems regarding these preceding studies in that subjects with different sized lesions and periods of time after lesioning were investigated; furthermore, the ability of blindsight was assessed with different measures. In this study, we used double dissociation to clarify the roles of the pulvinar and LGN by pharmacological inactivation of each region and investigated the effects in a simple task with visually guided saccades (VGSs) using monkeys with a unilateral V1 lesion, by which nearly all of the contralesional visual field was affected. Inactivating either the ipsilesional pulvinar or LGN impaired VGS toward a visual stimulus in the affected field. In contrast, inactivation of the contralesional pulvinar had no clear effect, but inactivation of the contralesional LGN impaired VGS to the intact visual field. These results suggest that the pulvinar and LGN play key roles in performing the simple VGS task after V1 lesioning, and that the visuomotor functions of blindsight monkeys were supported by plastic changes in the visual pathway involving the pulvinar, which emerged after V1 lesioning.SIGNIFICANCE STATEMENT Many studies have been devoted to understanding the mechanism of mysterious symptom called "blindsight," in which patients with damage to the primary visual cortex (V1) can respond to visual stimuli despite loss of visual awareness. However, there is still a debate on the thalamic relay of visual signals. In this study, to pin down the issue, we tried double dissociation in the same subjects (hemi-blindsight macaque monkeys) and clarified that the lateral geniculate nucleus (LGN) plays a major role in simple visually guided saccades in the intact state, while both pulvinar and LGN critically contribute after the V1 lesioning, suggesting that plasticity in the visual pathway involving the pulvinar underlies the blindsight.

Properties of visually guided saccadic behavior and bottom-up attention in marmoset, macaque, and human.

Saccades are stereotypic behaviors whose investigation improves our understanding of how primate brains implement precise motor control. Furthermore, saccades offer an important window into the cognitive and attentional state of the brain. Historically, saccade studies have largely relied on macaques. However, the cortical network giving rise to the saccadic command is difficult to study in macaques because relevant cortical areas lie in deep sulci and are difficult to access. Recently, a New World monkey. the marmoset, has garnered attention as an alternative to macaques because of advantages including its smooth cortical surface. However, adoption of the marmoset for oculomotor research has been limited due to a lack of in-depth descriptions of marmoset saccade kinematics and their ability to perform psychophysical tasks. Here, we directly compare free-viewing and visually guided behavior of marmoset, macaque, and human engaged in identical tasks under similar conditions. In the video free-viewing task, all species exhibited qualitatively similar saccade kinematics up to 25° in amplitude although with different parameters. Furthermore, the conventional bottom-up saliency model predicted gaze targets at similar rates for all species. We further verified their visually guided behavior by training them with step and gap saccade tasks. In the step paradigm, marmosets did not show shorter saccade reaction time for upward saccades whereas macaques and humans did. In the gap paradigm, all species showed similar gap effect and express saccades. Our results suggest that the marmoset can serve as a model for oculomotor, attentional, and cognitive research while we need to be aware of their difference from macaque or human.NEW & NOTEWORTHY We directly compared the results of a video free-viewing task and visually guided saccade tasks (step and gap) among three different species: marmoset, macaque, and human. We found that all species exhibit qualitatively similar saccadic kinematics and saliency-driven saccadic behavior albeit with different parameters. Our results suggest that the marmoset possesses similar neural mechanisms to macaque and human for saccadic control, and it is an appropriate model to study neural mechanisms for active vision and attention.

Enhanced Retrieval of Taste Associative Memory by Chemogenetic Activation of Locus Coeruleus Norepinephrine Neurons.

The ability of animals to retrieve memories stored in response to the environment is essential for behavioral adaptation. Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation. However, the role of the central NE system in memory retrieval remains unclear. Here, we developed a novel chemogenetic activation strategy exploiting insect olfactory ionotropic receptors (IRs), termed "IR-mediated neuronal activation," and used it for selective stimulation of NE neurons in the locus coeruleus (LC). Drosophila melanogaster IR84a and IR8a subunits were expressed in LC NE neurons in transgenic mice. Application of phenylacetic acid (a specific ligand for the IR84a/IR8a complex) at appropriate doses induced excitatory responses of NE neurons expressing the receptors in both slice preparations and in vivo electrophysiological conditions, resulting in a marked increase of NE release in the LC nerve terminal regions (male and female). Ligand-induced activation of LC NE neurons enhanced the retrieval process of conditioned taste aversion without affecting taste sensitivity, general arousal state, and locomotor activity. This enhancing effect on taste memory retrieval was mediated, in part, through α1- and ß-adrenergic receptors in the basolateral nucleus of the amygdala (BLA; male). Pharmacological inhibition of LC NE neurons confirmed the facilitative role of these neurons in memory retrieval via adrenergic receptors in the BLA (male). Our findings indicate that the LC NE system, through projections to the BLA, controls the retrieval process of taste associative memory.SIGNIFICANCE STATEMENT Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation, but the role of the NE system in memory retrieval remains unclear. We developed a chemogenetic activation system based on insect olfactory ionotropic receptors and used it for selective stimulation of NE neurons in the locus coeruleus (LC) in transgenic mice. Ligand-induced activation of LC NE neurons enhanced the retrieval of conditioned taste aversion, which was mediated, in part, through adrenoceptors in the basolateral amygdala. Pharmacological blockade of LC activity confirmed the facilitative role of these neurons in memory retrieval. Our findings indicate that the LC-amygdala pathway plays an important role in the recall of taste associative memory.

Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm3 tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.