Reconstruction Of Neuronal Networks Research Articles

Transplantation of Pre‐Differentiated 3D Neural Spheroids in Decellularized Extracellular Matrix Microgels Promotes Neuronal Network Reconstruction and Functional Recovery after Severe Spinal Cord Contusion

AbstractTraumatic spinal cord injury (SCI) causes massive death of neurons in the spinal cords and almost complete neurological dysfunctions. Transplantation of neural stem/progenitor cells (NSPCs) is acknowledged as one of the viable SCI treatments for complementing lost neurons and neural network reconstruction. However, administration of NSPCs suffers from extremely low survival rate and uncontrolled differentiation of the transplanted cells, which impairs the therapeutic effects significantly. Herein, NSPCs are encapsulated in decellularized spinal cord matrix (DSCM) microgels using a customized microfluidic system, then the obtained NSPCs‐encapsulated DSCM microgels (NSPC@DSCM‐MGs) are subjected to neuronal differentiation induction. Consequently, the resulting pre‐mature 3D neural spheroids are injected into severely contused spinal cords in rats. The DSCM microgels effectively protected the transplanted cells from shear damage and the inflammatory microenvironment at the lesion site. The survival and accommodation of the pre‐differentiated NSPC@DSCM‐MGs actively contributed to axonal regeneration, inhibiting glial scar formation, as well as remodeling the microenvironment that facilitated endogenous cell recruitment and neuronal network reconstruction. Consequentially, administration of the neural spheroids led to maintenance of spinal cord integrity, and significantly improved hindlimb locomotor function. This biomaterial‐based transplantation strategy has shown unique assets in cell protection and cell‐fate manipulation, which holds great promise in versatile biomedical applications.

Updated Toolbox for Assessing Neuronal Network Reconstruction after Cell Therapy.

Cell therapy has proven to be a promising treatment for a range of neurological disorders, including Parkinson Disease, drug-resistant epilepsy, and stroke, by restoring function after brain damage. Nevertheless, evaluating the true effectiveness of these therapeutic interventions requires a deep understanding of the functional integration of grafted cells into existing neural networks. This review explores a powerful arsenal of molecular techniques revolutionizing our ability to unveil functional integration of grafted cells within the host brain. From precise manipulation of neuronal activity to pinpoint the functional contribution of transplanted cells by using opto- and chemo-genetics, to real-time monitoring of neuronal dynamics shedding light on functional connectivity within the reconstructed circuits by using genetically encoded (calcium) indicators in vivo. Finally, structural reconstruction and mapping communication pathways between grafted and host neurons can be achieved by monosynaptic tracing with viral vectors. The cutting-edge toolbox presented here holds immense promise for elucidating the impact of cell therapy on neural circuitry and guiding the development of more effective treatments for neurological disorders.

A Compartmentalized Neuronal Cell-Culture Platform Compatible With Cryo-Fixation by High-Pressure Freezing for Ultrastructural Imaging.

The human brain contains a wide array of billions of neurons and interconnections, which are often simplified for analysis in vitro using compartmentalized microfluidic devices for neuronal cell culturing, to better understand neuronal development and disease. However, such devices are traditionally incompatible for high-pressure freezing and high-resolution nanoscale imaging and analysis of their sub-cellular processes by methods including electron microscopy. Here we develop a novel compartmentalized neuronal co-culture platform allowing reconstruction of neuronal networks with high variable spatial control, which is uniquely compatible for high-pressure freezing. This cryo-fixation method is well-established to enable high-fidelity preservation of the reconstructed neuronal networks and their sub-cellular processes in a near-native vitreous state without requiring chemical fixatives. To direct the outgrowth of neurites originating from two distinct groups of neurons growing in the two different compartments, polymer microstructures akin to microchannels are fabricated atop of sapphire disks. Two populations of neurons expressing either enhanced green fluorescent protein (EGFP) or mCherry were grown in either compartment, facilitating the analysis of the specific interactions between the two separate groups of cells. Neuronally differentiated PC12 cells, murine hippocampal and striatal neurons were successfully used in this context. The design of this device permits direct observation of entire neuritic processes within microchannels by optical microscopy with high spatial and temporal resolution, prior to processing for high-pressure freezing and electron microscopy. Following freeze substitution, we demonstrate that it is possible to process the neuronal networks for ultrastructural imaging by electron microscopy. Several key features of the embedded neuronal networks, including mitochondria, synaptic vesicles, axonal terminals, microtubules, with well-preserved ultrastructures were observed at high resolution using focused ion beam – scanning electron microscopy (FIB-SEM) and serial sectioning – transmission electron microscopy (TEM). These results demonstrate the compatibility of the platform with optical microscopy, high-pressure freezing and electron microscopy. The platform can be extended to neuronal models of brain disease or development in future studies, enabling the investigation of subcellular processes at the nanoscale within two distinct groups of neurons in a functional neuronal pathway, as well as pharmacological testing and drug screening.

UNI-EM: An Environment for Deep Neural Network-Based Automated Segmentation of Neuronal Electron Microscopic Images

Recently, there has been rapid expansion in the field of micro-connectomics, which targets the three-dimensional (3D) reconstruction of neuronal networks from stacks of two-dimensional (2D) electron microscopy (EM) images. The spatial scale of the 3D reconstruction increases rapidly owing to deep convolutional neural networks (CNNs) that enable automated image segmentation. Several research teams have developed their own software pipelines for CNN-based segmentation. However, the complexity of such pipelines makes their use difficult even for computer experts and impossible for non-experts. In this study, we developed a new software program, called UNI-EM, for 2D and 3D CNN-based segmentation. UNI-EM is a software collection for CNN-based EM image segmentation, including ground truth generation, training, inference, postprocessing, proofreading, and visualization. UNI-EM incorporates a set of 2D CNNs, i.e., U-Net, ResNet, HighwayNet, and DenseNet. We further wrapped flood-filling networks (FFNs) as a representative 3D CNN-based neuron segmentation algorithm. The 2D- and 3D-CNNs are known to demonstrate state-of-the-art level segmentation performance. We then provided two example workflows: mitochondria segmentation using a 2D CNN and neuron segmentation using FFNs. By following these example workflows, users can benefit from CNN-based segmentation without possessing knowledge of Python programming or CNN frameworks.

Development of New Therapies for Neurodegenerative Diseases via Axonal Growth

In neurodegenerative diseases, such as Alzheimer's disease (AD) and spinal cord injury (SCI), inhibited axonal regeneration lead to irreversible functional impairment. Although many agents that eliminate axonal growth impediments have been clinically investigated, none induced functional recovery. I hypothesized that the removal of impediments alone was not enough and that promoting axonal growth and neuronal network reconstruction were needed for recovery from neurodegenerative diseases. To promote axonal growth, I have focused on neurons and microglia. In vitro models of AD and SCI were developed by culturing neurons in the presence of amyloid β (Aβ) and chondroitin sulfate proteoglycan, respectively. These were then used to identify several extracts of herbal medicines and their constituents that promoted axonal growth. Oral administration of these extracts and their constituents improved memory and motor function in in vivo mouse models of AD and SCI, respectively. The bioactive compounds in these extracts were identified by analyzing brain and spinal cord samples from the mice. Their protein targets were identified using the drug affinity responsive target stability method. Analysis of early events in the axons after culture with Aβ revealed that the inhibition of endocytosis was sufficient to prevent the axonal atrophy and memory deficits caused by Aβ. The compounds that increased M2 microglia were observed to promote axonal normalization and growth; they were also found to recover memory and motor function in mice models of AD and SCI, respectively. The above results indicate that axonal growth plays important roles in the recovery from AD and SCI.

Identification of Neuronal Networks from Calcium Oscillation Data

Abstract Neuronal network reconstruction is an important problem in neuroscience as it helps understanding neuronal circuit and function. With the advancements of the calcium imaging technique, the dynamic activity of hundreds of neurons can be observed, which also provides a foundation for modelling and inferring network connectivity directly from data. In this paper, the dynamic behavior of each neuron is described using a non-linear autoregressive exogenous input (NARX) model. Since the neurons are inter-connected, the NARX models contain inputs which depend on outputs of the surrounding neurons. The complete model is expressed in a linear-in-parameter form and the network dynamics are identified by obtaining a block-sparse solution given the calcium oscillation data. The solution to the identification problem also provides the network topology simultaneously, i.e., the physical connections between the neurons. To demonstrate the accuracy of the proposed method, two experimental case studies are considered and the results are compared with other network identification methods.

Exploiting natural polysaccharides to enhance in vitro bio-constructs of primary neurons and progenitor cells

Current strategies in Central Nervous System (CNS) repair focus on the engineering of artificial scaffolds for guiding and promoting neuronal tissue regrowth. Ideally, one should combine such synthetic structures with stem cell therapies, encapsulating progenitor cells and instructing their differentiation and growth. We used developments in the design, synthesis, and characterization of polysaccharide-based bioactive polymeric materials for testing the ideal composite supporting neuronal network growth, synapse formation and stem cell differentiation into neurons and motor neurons. Moreover, we investigated the feasibility of combining these approaches with engineered mesenchymal stem cells able to release neurotrophic factors. We show here that composite bio-constructs made of Chitlac, a Chitosan derivative, favor hippocampal neuronal growth, synapse formation and the differentiation of progenitors into the proper neuronal lineage, that can be improved by local and continuous delivery of neurotrophins. Statement of SignificanceIn our work, we characterized polysaccharide-based bioactive platforms as biocompatible materials for nerve tissue engineering. We show that Chitlac-thick substrates are able to promote neuronal growth, differentiation, maturation and formation of active synapses. These observations support this new material as a promising candidate for the development of complex bio-constructs promoting central nervous system regeneration. Our novel findings sustain the exploitation of polysaccharide-based scaffolds able to favour neuronal network reconstruction. Our study shows that Chitlac-thick may be an ideal candidate for the design of biomaterial scaffolds enriched with stem cell therapies as an innovative approach for central nervous system repair.

Trigonelline promotes auditory function through nerve growth factor signaling on diabetic animal models

BackgroundProtection of cochlear function and reconstruction of neuronal networks in damaged auditory sensory structures is crucial for therapeutic treatment of diabetic hearing loss. Nerve growth factor (NGF) has been used as a novel therapeutic target to protect against the neurodegenerative effects of Diabetes Mellitus (DM). PurposeWe aimed to evaluate the potential effect of trigonelline (TRG) on reducing auditory damage produced by DM using NGF as a potential marker. MethodDocking simulations were carried out using Autodock Vina software and visualized using Discovery Studio. Morphological analysis of hair cells and neuromasts was performed on alloxan-induced diabetic zebrafish by fluorescence and scanning electron microscopy. Blockage of NGF receptor phosphorylation with K-252a was used to evaluate TRG and NGF action. Further assessment of NGF by ELISA on a primary culture of spiral ganglion cells was performed as a marker of neuronal function on the hearing system. Finally, auditory function was assessed in LepR(db/db) mice using auditory brainstem response (ABR) and transient evoked otoacoustic emission (TEOAE) during 8 weeks. ResultsDocking simulations showed that TRG binds to the active site of NGF through molecular interactions with Lysine88 (Lys88) and Tyrosine52 (Tyr52). TRG treatment significantly reduced hair cell loss and neuromast damage in diabetic zebrafish (P < .05). Further evaluation revealed a significant increase in the number of neuromasts after NGF administration (P < .001). TRG and NGF action was suppressed during blockage of NGF receptor phosphorylation. Moreover, spiral ganglion cells revealed significant elevation on NGF values after TRG treatment (P < .05). In vivo evaluation of LepR(db/db) mice revealed a significant reduction in the auditory damage produced under diabetic progression, characterized by reduced ABR hearing threshold shifts and increased signal-to-noise ratio in TEOAE (P < .05). ConclusionsThis study suggests that the enhanced hearing function produced by TRG may be mediated by NGF, providing a potential therapeutic strategy for diabetic hearing loss.

Slit2/Robo1 promotes synaptogenesis and functional recovery of spinal cord injury.

Neuronal network reconstruction is a pivotal determinant for functional recovery after spinal cord injury (SCI), the process of which includes synaptogenesis. Slit2 protein has been identified as a key regulator of axon regeneration and synapse formation in the vertebrate. Meanwhile, RhoA is the converging cascade of inhibitory molecules that interrupt synaptic plasticity in SCI. In the present study, we investigated the interaction among Slit2, Robo1, and RhoA and the potential roles of Slit2 in the pathological process of SCI. We showed that Slit2 was decreased, whereas Robo1 and RhoA were increased in the same surviving neurons in the spinal cord following SCI. We also found that inhibition of Slit2 led to upregulation of the expression of Robo1 and RhoA. However, the severe dysfunctions of the locomotor performance induced by SCI were reversed by treatments of Slit2-N, the active portion of Slit2, knockdown of Robo1 by the RNAi lentivirus, or inhibition of RhoA by the C3 exoenzyme, respectively. Further results suggested that downregulation of Slit2 and therefore upregulation of Robo1 and RhoA inhibited the activity of growth cone and hindered the formation of new synapses of surviving neurons near the injury sites of the spinal cord following SCI. Our study indicated a new mechanism of deficiency of synaptogenesis during the development of SCI and provided a potential strategy for the treatment of SCI.

Transplantation of Embryonic Cortical Tissue into Lesioned Adult Brain in Mice.

Transplantation of embryonic cortical tissue for repairing the damaged brain has provided a potential therapy for brain injury and diseases. The grafted tissue can successfully survive and participate in reestablishing the functional neural circuit of the host brain. Transplantation surgery can be combined with fluorescently labeled transgenic mice to evaluate the reconstruction of neuronal network ( Falkner et al., 2016 ) and the repopulation of a subset of cortical cells. By using this approach, we have shown that infiltrating cells from host brain can restore the microglial population in the graft tissue ( Wang et al., 2016 ). This protocol describes the detailed procedure of the transplantation surgery in mice, including establishing a lesion model in the host brain, preparing the embryonic cortical graft, and transplanting the embryonic cortical graft to adult brain.

In Vitro Reconstruction of Neuronal Networks Derived from Human iPS Cells Using Microfabricated Devices.

Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues, which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases, appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study, we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS), or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally, calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next, we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs, and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions.

New Age Therapy for Alzheimer's Disease by Neuronal Network Reconstruction.

Alzheimer's disease (AD) is a recognized incurable neurodegenerative disorder. Clinically prescribed medicines for AD are expected to bring about only slight symptomatic improvement or a delay of its progression. Another strategy, amyloid β (Aβ) lowing agents, has not been successful at memory improvement. We have hypothesized that an improvement in cognitive function requires the construction of neuronal networks, including neurite regeneration and synapse formation; therefore, we have been exploring candidates for radical anti-AD drugs that can restore Aβ-induced neurite atrophy and memory impairment. Our studies found several promising drug candidates that may improve memory dysfunction in AD model mice. The main activity of these drugs is the restoration of damaged axons. Focusing on candidates based on the recovery of neurite atrophy in vitro certainly leads to positive effects on memory improvement also in vivo. This suggests that neuronal network reconstruction may importantly relate to functional recovery in the brain. When identifying the signaling mechanisms of exogenous compounds like natural medicine-derived constituents, molecules directly activated by the compound are hard to be identified. However, the drug affinity responsive target stability (DARTS) analysis may pave the way to an approach to determine the initial molecule of the signaling pathway. Exploring new drug candidates and clarifying their signaling pathways directly relating to neuronal network reconstruction may provide promising therapeutic strategies with which to overcome AD.

Methods for the analysis of neuronal plasticity and brain connectivity during neurological recovery.

The study of neuronal plasticity under pathological conditions is now a major point of focus on the field of neurological recovery. After the repeated failure of acute neuroprotection strategies for stroke treatment, the design of studies aimed at promoting the reconstruction of neuronal networks has become essential. Methods for the delivery of therapeutic agents on a steady dosage, thus preventing pharmacological peaks or excessive manipulation of experimental animals, are thus required. Additionally, methods that allow the visualization of neurological remodeling processes are fundamental to the understanding of how a therapeutic agent exerts its function. Here we describe how the use of miniosmotic pumps for the steady delivery of such agents, together with tract tracer injections, can be combined to unveil important information on how the brain changes after stroke and how therapeutic agents promote brain remodeling recovery.

A combination of keratan sulfate digestion and rehabilitation promotes anatomical plasticity after rat spinal cord injury

Functional recovery after neuronal injuries relies on neuronal network reconstruction which involves many repair processes, such as sealing of injured axon ends, axon regeneration/sprouting, and construction and refinement of synaptic connections. Chondroitin sulfate (CS) is a major inhibitor of axon regeneration/sprouting. It has been reported that the combination of task-specific rehabilitation and CS-digestion is much more effective than either treatment alone with regard to the promotion of functional and anatomical plasticity for dexterity in acute and chronic spinal cord injury models. We previously reported that keratan sulfate (KS) is another inhibitor and has a potency equal to CS. Here, we compared the effects of KS- or CS-digestion plus rehabilitation on recovery from spinal cord injury. Keratanase II or chondroitinase ABC was locally administered at the lesion after spinal cord injury at C3/4. Task-specific rehabilitation training, i.e., a single pellet reaching task using a Whishaw apparatus, was done for 3 weeks before injury, and then again at 1–6 weeks after injury. The combination of KS-digestion and rehabilitation yielded a better rate of pellet removal than either KS-digestion alone or rehabilitation alone, although these differences were not statistically significant. The combination of CS-digestion and rehabilitation showed similar results. Strikingly, both KS-digestion/rehabilitation and CS-digestion/rehabilitation showed significant increases in neurite growth in vivo as estimated by 5-hydroxytryptamine and GAP43 staining. Thus, KS-digestion and rehabilitation exerted a synergistic effect on anatomical plasticity, and this effect was comparable with that of CS-digestion/rehabilitation. KS-digestion might widen the therapeutic window of spinal cord injury if combined with rehabilitation.

Cell specific electrodes for neuronal network reconstruction and monitoring

Direct interfacing of neurons with electronic devices has been investigated for both prosthetic and neuro-computing applications. In vitro neuronal networks provide great tools not only for improving neuroprostheses but also to take advantage of their computing abilities. However, it is often difficult to organize neuronal networks according to specific cell distributions. Our aim was to develop a cell-type specific immobilization of neurons on individual electrodes to produce organized in vitro neuronal networks on multi-electrode arrays (MEAs). We demonstrate the selective capture of retinal neurons on antibody functionalized surfaces following the formation of self-assembled monolayers from protein-thiol conjugates by simple contact and protein-polypyrrole deposits by electrochemical functionalization. This neuronal selection was achieved on gold for either cone photoreceptors or retinal ganglion neurons using a PNA lectin or a Thy1 antibody, respectively. Anti-fouling of un-functionalized gold surfaces was optimized to increase the capture efficiencies. The technique was extended to electrode arrays by addressing electropolymerization of pyrrole monomers and pyrrole-protein conjugates to active electrodes. Retinal ganglion cell recording on the array further demonstrated the integrity of these neurons following their selection on polypyrrole-coated electrodes. Therefore, this protein-polypyrrole electrodeposition could provide a new approach to generate organized in vitro neuronal networks.

Transplantation of Bone Marrow Stromal Cell-Derived Neural Precursor Cells Ameliorates Deficits in a Rat Model of Complete Spinal Cord Transection

After severe spinal cord injury, spontaneous functional recovery is limited. Numerous studies have demonstrated cell transplantation as a reliable therapeutic approach. However, it remains unknown whether grafted neuronal cells could replace lost neurons and reconstruct neuronal networks in the injured spinal cord. To address this issue, we transplanted bone marrow stromal cell-derived neural progenitor cells (BM-NPCs) in a rat model of complete spinal cord transection 9 days after the injury. BM-NPCs were induced from bone marrow stromal cells (BMSCs) by gene transfer of the Notch-1 intracellular domain followed by culturing in the neurosphere method. As reported previously, BM-NPCs differentiated into neuronal cells in a highly selective manner in vitro. We assessed hind limb movements of the animals weekly for 7 weeks to monitor functional recovery after local injection of BM-NPCs to the transected site. To test the sensory recovery, we performed functional magnetic resonance imaging (fMRI) using electrical stimulation of the hind limbs. In the injured spinal cord, transplanted BM-NPCs were confirmed to express neuronal markers 7 weeks following the transplantation. Grafted cells successfully extended neurites beyond the transected portion of the spinal cord. Adjacent localization of synaptophysin and PSD-95 in the transplanted cells suggested synaptic formations. These results indicated survival and successful differentiation of BM-NPCs in the severely injured spinal cord. Importantly, rats that received BM-NPCs demonstrated significant motor recovery when compared to the vehicle injection group. Volumes of the fMRI signals in somatosensory cortex were larger in the BM-NPC-grafted animals. However, neuronal activity was diverse and not confined to the original hind limb territory in the somatosensory cortex. Therefore, reconstruction of neuronal networks was not clearly confirmed. Our results indicated BM-NPCs as an effective method to deliver neuronal lineage cells in a severely injured spinal cord. However, reestablishment of neuronal networks in completed transected spinal cord was still a challenging task.

Reconstruction of Neuronal Networks in the Central Nervous System with Nano-Biotechnology and Photoelectronic Tools

Reconstruction of Neuronal Networks in the Central Nervous System with Nano-Biotechnology and Photoelectronic Tools

Reconstruction of Neuronal Networks in vitro

Reconstruction of Neuronal Networks in vitro

Flat and tubular membrane systems for the reconstruction of hippocampal neuronal network

The selection of appropriate biomaterials that promote cellular adhesion and growth is particularly important for the in vitro reconstruction of neuronal network. This study focused on the development of new polymeric membranes in flat and tubular (hollow-fibre) configurations as novel biomaterials for neuronal outgrowth. Two membrane systems constituted by modified polyetheretherketone (PEEK-WC) and polyacrylonitrile (PAN) membranes were developed and used for the culture of hamster hippocampal neurons. We demonstrated that all investigated membranes supported the adhesion and growth of hippocampal neurons enhancing neuronal differentiation and neurite alignment. The differences in cell behaviours between cells cultured on flat and hollow-fibre (HF) membranes were highlighted by the quantitative analysis of neuronal marker fluorescence intensity, morphometric analysis, RT-PCR analysis and also by metabolic activity measurements. In particular, the PAN HF membranes showed ideal growth culture conditions, guaranteeing adequate levels of metabolic features. Primary hippocampal cells cultured on PAN HF membranes were able to recreate in vitro a 3D neural tissue-like structure that, mimicking the hippocampal tissue, could be used as a tool for the study of natural and pathological neurobiological events.

Kihi-to, a herbal traditional medicine, improves Abeta(25–35)-induced memory impairment and losses of neurites and synapses

BackgroundWe previously hypothesized that achievement of recovery of brain function after the injury requires the reconstruction of neuronal networks, including neurite regeneration and synapse reformation. Kihi-to is composed of twelve crude drugs, some of which have already been shown to possess neurite extension properties in our previous studies. The effect of Kihi-to on memory deficit has not been examined. Thus, the goal of the present study is to determine the in vivo and in vitro effects of Kihi-to on memory, neurite growth and synapse reconstruction.MethodsEffects of Kihi-to, a traditional Japanese-Chinese traditional medicine, on memory deficits and losses of neurites and synapses were examined using Alzheimer's disease model mice. Improvements of Aβ(25–35)-induced neuritic atrophy by Kihi-to and the mechanism were investigated in cultured cortical neurons.ResultsAdministration of Kihi-to for consecutive 3 days resulted in marked improvements of Aβ(25–35)-induced impairments in memory acquisition, memory retention, and object recognition memory in mice. Immunohistochemical comparisons suggested that Kihi-to attenuated neuritic, synaptic and myelin losses in the cerebral cortex, hippocampus and striatum. Kihi-to also attenuated the calpain increase in the cerebral cortex and hippocampus. When Kihi-to was added to cells 4 days after Aβ(25–35) treatment, axonal and dendritic outgrowths in cultured cortical neurons were restored as demonstrated by extended lengths of phosphorylated neurofilament-H (P-NF-H) and microtubule-associated protein (MAP)2-positive neurites. Aβ(25–35)-induced cell death in cortical culture was also markedly inhibited by Kihi-to. Since NF-H, MAP2 and myelin basic protein (MBP) are substrates of calpain, and calpain is known to be involved in Aβ-induced axonal atrophy, expression levels of calpain and calpastatin were measured. Treatment with Kihi-to inhibited the Aβ(25–35)-evoked increase in the calpain level and decrease in the calpastatin level. In addition, Kihi-to inhibited Aβ(25–35)-induced calcium entry.ConclusionIn conclusion Kihi-to clearly improved the memory impairment and losses of neurites and synapses.