Megumi Eguchi

Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.

In vivo and in vitro visualization of gene expression dynamics over extensive areas of the brain

In vivo monitoring of gene expression using promoter-destabilized fluorescence protein constructs is a powerful method for examining the expression dynamics of immediate-early genes in the brain. However, weak fluorescence signals derived from such constructs have hampered analyses of gene expression over extensive areas of the brain. We succeeded in producing transgenic mice with brains exhibiting high level expression of the reporter gene driven by the Arc gene promoter, which is activated in association with various brain functions (reporter mRNA abundance was near 100-fold greater than endogenous Arc mRNAs). This high expression of the reporter gene enabled us to monitor Arc gene expression dynamics in vivo, over an area that included the whole of the dorsal cerebral cortex. Moreover, we were able to perform three-dimensional analyses of activated regions using paraformaldehyde-fixed brains. In addition to the visual cortex, we found that the cingulate cortex was strongly activated by light stimuli. These mice are extremely useful for the functional analysis of gene expression over extensive areas of the brains in both wild-type mice and mutants with impaired brain function.

Orchestrated experience-driven Arc responses are disrupted in a mouse model of Alzheimer's disease.

Experience-induced expression of immediate-early gene Arc (also known as Arg3.1) is known to be important for consolidation of memory. Using in vivo longitudinal multiphoton imaging, we found orchestrated activity-dependent expression of Arc in the mouse extrastriate visual cortex in response to a structured visual stimulation. In wild-type mice, the amplitude of the Arc response in individual neurons strongly predicted the probability of reactivation by a subsequent presentation of the same stimulus. In a mouse model of Alzheimers disease, this association was markedly disrupted in the cortex, specifically near senile plaques. Neurons in the vicinity of plaques were less likely to respond, but, paradoxically, there were stronger responses in those few neurons around plaques that did respond. To the extent that the orchestrated pattern of Arc expression reflects nervous system responses to and physiological consolidation of behavioral experience, the disruption in Arc patterns reveals plaque-associated interference with neural network integration.

Neural activity changes underlying the working memory deficit in alpha-CaMKII heterozygous knockout mice

The alpha-isoform of calcium/calmodulin-dependent protein kinase II (α-CaMKII) is expressed abundantly in the forebrain and is considered to have an essential role in synaptic plasticity and cognitive function. Previously, we reported that mice heterozygous for a null mutation of α-CaMKII (α-CaMKII+/−) have profoundly dysregulated behaviors including a severe working memory deficit, which is an endophenotype of schizophrenia and other psychiatric disorders. In addition, we found that almost all the neurons in the dentate gyrus (DG) of the mutant mice failed to mature at molecular, morphological and electrophysiological levels. In the present study, to identify the brain substrates of the working memory deficit in the mutant mice, we examined the expression of the immediate early genes (IEGs), c-Fos and Arc, in the brain after a working memory version of the eight-arm radial maze test. c-Fos expression was abolished almost completely in the DG and was reduced significantly in neurons in the CA1 and CA3 areas of the hippocampus, central amygdala, and medial prefrontal cortex (mPFC). However, c-Fos expression was intact in the entorhinal and visual cortices. Immunohistochemical studies using arc promoter driven dVenus transgenic mice demonstrated that arc gene activation after the working memory task occurred in mature, but not immature neurons in the DG of wild-type mice. These results suggest crucial insights for the neural circuits underlying spatial mnemonic processing during a working memory task and suggest the involvement of α-CaMKII in the proper maturation and integration of DG neurons into these circuits.

Right-hemispheric dominance of spatial memory in split-brain mice.

Left‐right asymmetry of human brain function has been known for a century, although much of molecular and cellular basis of brain laterality remains to be elusive. Recent studies suggest that hippocampal CA3‐CA1 excitatory synapses are asymmetrically arranged, however, the functional implication of the asymmetrical circuitry has not been studied at the behavioral level. In order to address the left‐right asymmetry of hippocampal function in behaving mice, we analyzed the performance of “split‐brain” mice in the Barnes maze. The “split‐brain” mice received ventral hippocampal commissure and corpus callosum transection in addition to deprivation of visual input from one eye. In such mice, the hippocampus in the side of visual deprivation receives sensory‐driven input. Better spatial task performance was achieved by the mice which were forced to use the right hippocampus than those which were forced to use the left hippocampus. In two‐choice spatial maze, forced usage of left hippocampus resulted in a comparable performance to the right counterpart, suggesting that both hippocampal hemispheres are capable of conducting spatial learning. Therefore, the results obtained from the Barnes maze suggest that the usage of the right hippocampus improves the accuracy of spatial memory. Performance of non‐spatial yet hippocampus‐dependent tasks (e.g. fear conditioning) was not influenced by the laterality of the hippocampus.

Synaptic Plasticity Associated with a Memory Engram in the Basolateral Amygdala

Synaptic plasticity is a cellular mechanism putatively underlying learning and memory. However, it is unclear whether learning induces synaptic modification globally or only in a subset of neurons in associated brain regions. In this study, we genetically identified neurons activated during contextual fear learning and separately recorded synaptic efficacy from recruited and nonrecruited neurons in the mouse basolateral amygdala (BLA). We found that the fear learning induces presynaptic potentiation, which was reflected by an increase in the miniature EPSC frequency and by a decrease in the paired-pulse ratio. Changes occurred only in the cortical synapses targeting the BLA neurons that were recruited into the fear memory trace. Furthermore, we found that fear learning reorganizes the neuronal ensemble responsive to the conditioning context in conjunction with the synaptic plasticity. In particular, the neuronal activity during learning was associated with the neuronal recruitment into the context-responsive ensemble. These findings suggest that synaptic plasticity in a subset of BLA neurons contributes to fear memory expression through ensemble reorganization.

Visualization of Arc promoter-driven neuronal activity by magnetic resonance imaging

Visualization of direct neuronal activity to understand brain function is one of the most important challenges in neuroscience. We have previously demonstrated that in vivo and in vitro gene expression of the ferritin reporter system could be detected by magnetic resonance imaging (MRI). In addition, increased neuronal activity induces Arc, an immediate early gene, and insertion of a destabilized fluorescent reporter dVenus under Arc promoter control has been used for monitoring neuronal activities in the brain by optical imaging. In this study, to visualize Arc promoter-driven neuronal activities directly, we generated transgenic mice and cell lines that express a destabilized fusion reporter ferritin-mKate2 under Arc promoter control. When transgenic mice and cell lines were treated with pilocarpine, a non-selective muscarinic agonist, an increase in T2-weighted image signal was successfully found in neuronal cells. There was a difference in peak time between MRI and fluorescence imaging, which might result from the binding process of iron with ferritin. Visualization of Arc promoter-driven neuronal activity is essential to understand neural mechanisms underlying cognitive processes and complex behaviors, and could be a useful tool for therapeutic approaches in the brain by MRI.

LC-MS/MS imaging with thermal film-based laser microdissection

AbstractMass spectrometry (MS) imaging is a useful tool for direct and simultaneous visualization of specific molecules. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to evaluate the abundance of molecules in tissues using sample homogenates. To date, however, LC-MS/MS has not been utilized as an imaging tool because spatial information is lost during sample preparation. Here we report a new approach for LC-MS/MS imaging using a thermal film-based laser microdissection (LMD) technique. To isolate tissue spots, our LMD system uses a 808-nm near infrared laser, the diameter of which can be freely changed from 2.7 to 500 μm; for imaging purposes in this study, the diameter was fixed at 40 μm, allowing acquisition of LC-MS/MS images at a 40-μm resolution. The isolated spots are arranged on a thermal film at 4.5-mm intervals, corresponding to the well spacing on a 384-well plate. Each tissue spot is handled on the film in such a manner as to maintain its spatial information, allowing it to be extracted separately in its individual well. Using analytical LC-MS/MS in combination with the spatial information of each sample, we can reconstruct LC-MS/MS images. With this imaging technique, we successfully obtained the distributions of pilocarpine, glutamate, γ-aminobutyric acid, acetylcholine, and choline in a cross-section of mouse hippocampus. The protocol we established in this study is applicable to revealing the neurochemistry of pilocarpine model of epilepsy. Our system has a wide range of uses in fields such as biology, pharmacology, pathology, and neuroscience. Graphical abstractSchematic Indication of LMD-LC-MS/MS imaging.

Common Hepatic Branch of Vagus Nerve-Dependent Expression of Immediate Early Genes in the Mouse Brain by Intraportal L-Arginine: Comparison with Cholecystokinin-8

Information from the peripheral organs is thought to be transmitted to the brain by humoral factors and neurons such as afferent vagal or spinal nerves. The common hepatic branch of the vagus (CHBV) is one of the main vagus nerve branches, and consists of heterogeneous neuronal fibers that innervate multiple peripheral organs such as the bile duct, portal vein, paraganglia, and gastroduodenal tract. Although, previous studies suggested that the CHBV has a pivotal role in transmitting information on the status of the liver to the brain, the details of its central projections remain unknown. The purpose of the present study was to investigate the brain regions activated by the CHBV. For this purpose, we injected L-arginine or anorexia-associated peptide cholecystokinin-8 (CCK), which are known to increase CHBV electrical activity, into the portal vein of transgenic Arc-dVenus mice expressing the fluorescent protein Venus under control of the activity-regulated cytoskeleton-associated protein (Arc) promotor. The brain slices were prepared from these mice and the number of Venus positive cells in the slices was counted. After that, c-Fos expression in these slices was analyzed by immunohistochemistry using the avidin-biotin-peroxidase complex method. Intraportal administration of L-arginine increased the number of Venus positive or c-Fos positive cells in the insular cortex. This action of L-arginine was not observed in CHBV-vagotomized Arc-dVenus mice. In contrast, intraportal administration of CCK did not increase the number of c-Fos positive or Venus positive cells in the insular cortex. Intraportal CCK induced c-Fos expression in the dorsomedial hypothalamus, while intraportal L-arginine did not. This action of CCK was abolished by CHBV vagotomy. Intraportal L-arginine reduced, while intraportal CCK increased, the number of c-Fos positive cells in the nucleus tractus solitarii in a CHBV-dependent manner. The present results suggest that the CHBV can activate different brain regions depending on the nature of the peripheral stimulus.

Fear conditioning increases transmitter release to specific subsets of basolateral amygdala neurons

group, n = 11), (C) participants who did not receive the feedback with the praise (control group, n = 15). On the next day, all participants performed a surprise recall test of the learned sequence. The recall performance was compared among the three groups. The performance was defined as the maximal number of sequence they performed correctly during 30 seconds. In the all groups, the recall performance was significantly improved compared to the performance at the end of the training day (p < .001, for all group), confirming the off-line improvement, a form of consolidation (Robertson et al., 2004). However, the rate of the off-line improvement was significantly greater in the self-praised group compared to the other-praised (p < .05) or control groups (p < .01). The performances of the non-learned sequential or random-ordered finger-tapping tasks were not significantly different among the groups. These results suggest that social reward after the procedural motor training could facilitate its off-line improvement. Research fund: KAKENHI (22700442).