Naoki Matsuo

Localization of a Stable Neural Correlate of Associative Memory

Do learning and retrieval of a memory activate the same neurons? Does the number of reactivated neurons correlate with memory strength? We developed a transgenic mouse that enables the long-lasting genetic tagging of c-fos–active neurons. We found neurons in the basolateral amygdala that are activated during Pavlovian fear conditioning and are reactivated during memory retrieval. The number of reactivated neurons correlated positively with the behavioral expression of the fear memory, indicating a stable neural correlate of associative memory. The ability to manipulate these neurons genetically should allow a more precise dissection of the molecular mechanisms of memory encoding within a distributed neuronal network.

Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum

The molecular machinery governing glutamatergic-GABAergic neuronal subtype specification is unclear. Here we describe a cerebellar mutant, cerebelless, which lacks the entire cerebellar cortex in adults. The primary defect of the mutant brains was a specific inhibition of GABAergic neuron production from the cerebellar ventricular zone (VZ), resulting in secondary and complete loss of external germinal layer, pontine, and olivary nuclei during development. We identified the responsible gene, Ptf1a, whose expression was lost in the cerebellar VZ but was maintained in the pancreas in cerebelless. Lineage tracing revealed that two types of neural precursors exist in the cerebellar VZ: Ptf1a-expressing and -nonexpressing precursors, which generate GABAergic and glutamatergic neurons, respectively. Introduction of Ptf1a into glutamatergic neuron precursors in the dorsal telencephalon generated GABAergic neurons with representative morphological and migratory features. Our results suggest that Ptf1a is involved in driving neural precursors to differentiate into GABAergic neurons in the cerebellum.

Locally Synchronized Synaptic Inputs

Coordinating Synapses Cortical microcircuits produce cell assemblies that emit spatiotemporally orchestrated spiking activity. These activity patterns are decoded by the dendrites of downstream neurons. Whether synaptic inputs are clustered or dispersed over target dendrites at a given time is critical for determining dendritic computational power. However, such subcellular dynamics are poorly understood. In rodent organotypic slice cultures, Takahashi et al. (p. 353) found that dendritic spine activities were frequently synchronized within a group of spines in the immediate vicinity of one another. This local synchronization seems to reflect convergent synaptic inputs from intrinsically synchronized presynaptic neuron populations. The computational power of individual neurons is determined by a surprisingly precise wiring of neuronal networks. Synaptic inputs on dendrites are nonlinearly converted to action potential outputs, yet the spatiotemporal patterns of dendritic activation remain to be elucidated at single-synapse resolution. In rodents, we optically imaged synaptic activities from hundreds of dendritic spines in hippocampal and neocortical pyramidal neurons ex vivo and in vivo. Adjacent spines were frequently synchronized in spontaneously active networks, thereby forming dendritic foci that received locally convergent inputs from presynaptic cell assemblies. This precise subcellular geometry manifested itself during N-methyl-d-aspartate receptor–dependent circuit remodeling. Thus, clustered synaptic plasticity is innately programmed to compartmentalize correlated inputs along dendrites and may reify nonlinear synaptic integration.

Behavioral Profiles of Three C57BL/6 Substrains

C57BL/6 inbred strains of mice are widely used in knockout and transgenic research. To evaluate the loss-of-function and gain-of-function effects of the gene of interest, animal behaviors are often examined. However, an issue of C57BL/6 substrains that is not always appreciated is that behaviors are known to be strongly influenced by genetic background. To investigate the behavioral characteristics of C57BL/6 substrains, we subjected C57BL/6J, C57BL/6N, and C57BL/6C mice to a behavior test battery. We performed both a regular scale analysis, in which experimental conditions were tightly controlled, and large-scale analysis from large number of behavioral data that we have collected so far through the comprehensive behavioral test battery applied to 700–2,200 mice in total. Significant differences among the substrains were found in the results of various behavioral tests, including the open field, rotarod, elevated plus maze, prepulse inhibition, Porsolt forced swim, and spatial working memory version of the eight-arm radial maze. Our results show a divergence of behavioral performance in C57BL/6 substrains, which suggest that small genetic differences may have a great influence on behavioral phenotypes. Thus, the genetic background of different substrains should be carefully chosen, equated, and considered in the interpretation of mutant behavioral phenotypes.

Abnormal social behavior, hyperactivity, impaired remote spatial memory, and increased D1-mediated dopaminergic signaling in neuronal nitric oxide synthase knockout mice

BackgroundNeuronal nitric oxide synthase (nNOS) is involved in the regulation of a diverse population of intracellular messenger systems in the brain. In humans, abnormal NOS/nitric oxide metabolism is suggested to contribute to the pathogenesis and pathophysiology of some neuropsychiatric disorders, such as schizophrenia and bipolar disorder. Mice with targeted disruption of the nNOS gene exhibit abnormal behaviors. Here, we subjected nNOS knockout (KO) mice to a battery of behavioral tests to further investigate the role of nNOS in neuropsychiatric functions. We also examined the role of nNOS in dopamine/DARPP-32 signaling in striatal slices from nNOS KO mice and the effects of the administration of a dopamine D1 receptor agonist on behavior in nNOS KO mice.ResultsnNOS KO mice showed hyperlocomotor activity in a novel environment, increased social interaction in their home cage, decreased depression-related behavior, and impaired spatial memory retention. In striatal slices from nNOS KO mice, the effects of a dopamine D1 receptor agonist, SKF81297, on the phosphorylation of DARPP-32 and AMPA receptor subunit GluR1 at protein kinase A sites were enhanced. Consistent with the biochemical results, intraperitoneal injection of a low dose of SKF81297 significantly decreased prepulse inhibition in nNOS KO mice, but not in wild-type mice.ConclusionThese findings indicate that nNOS KO upregulates dopamine D1 receptor signaling, and induces abnormal social behavior, hyperactivity and impaired remote spatial memory. nNOS KO mice may serve as a unique animal model of psychiatric disorders.

Comprehensive Behavioral Phenotyping of Ryanodine Receptor type 3 (RyR3) Knockout Mice: Decreased Social Contact Duration in Two Social Interaction Tests

Dynamic regulation of the intracellular Ca2+ concentration is crucial for various neuronal functions such as synaptic transmission and plasticity, and gene expression. Ryanodine receptors (RyRs) are a family of intracellular calcium release channels that mediate calcium-induced calcium release from the endoplasmic reticulum. Among the three RyR isoforms, RyR3 is preferentially expressed in the brain especially in the hippocampus and striatum. To investigate the behavioral effects of RyR3 deficiency, we subjected RyR3 knockout (RyR3–/–) mice to a battery of behavioral tests. RyR3–/– mice exhibited significantly decreased social contact duration in two different social interaction tests, where two mice can freely move and make contacts with each other. They also exhibited hyperactivity and mildly impaired prepulse inhibition and latent inhibition while they did not show significant abnormalities in motor function and working and reference memory tests. These results indicate that RyR3 has an important role in locomotor activity and social behavior.

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.

Dynamic and coordinated expression profile of dbl-family guanine nucleotide exchange factors in the developing mouse brain

Dbl-family guanine nucleotide exchange factors (Dbl-GEFs) act as activators of Rho-like small G proteins such as Rac1, Cdc42 and RhoA. Recently, some GEFs have been suggested to play important roles in the development of the nervous system. Here, we report a comprehensive expression profile analysis of 20 Dbl-GEFs that have yet to be well investigated. Northern analyses of murine mRNAs from brains of E13, E17, P7 and adult mice revealed expression of 18 out of 20 GEFs in some or all stages. In addition, we found that three human GEFs were highly expressed in the brain. Examination of the spatial expression patterns of five GEFs in embryos or neonatal brain by in situ hybridization revealed distinct patterns for each GEF. Our study reveals the dynamic and coordinated expression profiles of the Dbl-GEFs and provides a basic framework for understanding the function of GEFs in neural development.

Behavioral profiles of three C57BL/6 substrains

s / Neuroscience Research 68S (2010) e335–e446 e433 P3-q06 Behavioral profiles of three C57BL/6 substrains Keizo Takao 1,2,3,4,5 , Naoki Matsuo 3,4, Kazuo Nakanishi 2,4,5, Nobuyuki Yamasaki 2,4,5, Koichi Tanda 2,4,5, Tsuyoshi Miyakawa 1,2,3,4,5 1 Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Japan 2 Frontier Technology Center, Graduate School of Medicine Kyoto University, Kyoto, Japan 3 Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan 4 JST, Core Research for Evolutional Science and Technology (CREST), Kawaguchi, Japan 5 JST, Institute for Bioinformatics Research and Development (BIRD), Kawaguchi, Japan C57BL/6 inbred strains of mice are widely used in knockout and transgenic research. To evaluate the loss-of-function and gain-of-function effects of the gene of interest, animal behaviors are often examined. However, an issue of C57BL/6 substrains is not always appreciated though behaviors of mice are known to be strongly influenced by genetic background. To investigate the behavioral characteristics of C57BL/6 substrains, we subjected C57BL/6J, C57BL/6N, and C57BL/6C mice to a behavioral test battery. We performed both a regular-scale analysis whose experimental conditions were tightly controlled and meta-analysis from large number of behavioral data, derived from 700∼2,200 mice in total, that we have collected so far through the comprehensive behavioral test battery. Significant differences were found in various behavioral tests, including the open field, rotarod, elevated plus maze, startle response/prepulse inhibition, Porsolt forced swim, and spatial working memory version of the 8-arm radial maze among the substrains. Our results show divergence of behavioral performance in C57BL/6 substrains and therefore indicate that small genetic differences might have great influence on behavioral phenotypes. Thus, genetic background of different substrains should be carefully chosen, equated, and considered in the interpretation of the mutant behavioral phenotypes. doi:10.1016/j.neures.2010.07.1918 P3-q07 Effects of multiple nerve crushes on functional recovery of the sciatic nerve Toshiro Itsubo 1 , Akira Kakegawa 2, Kumiko Yokouchi 2, Kyutaro Kawagishi 2, Tetsuji Moriizumi 2, Nanae Fukushima 2 1 Department of Orthopaedic Surgery, Shinshu University School of Medicine 2 Department of Anatomy, Shinshu University School of Medicine, Matsumoto, Japan Motor neurons can survive and reinnervate the muscles after the sciatic nerve crush injury. In this study, we investigated the effects of multiple nerve crush injuries on functional recovery and reinnervation of muscles. Using functional and immunohistochemical analyses, we evaluated the effects of nerve crush injuries. Motor function was estimated by the sciatic static index (SSI), measuring two parameters of 1-5 toe spread and 2–4 toe spread lengths on both sides, and the SSI scores became a normal range in 4 weeks after the single sciatic nerve crush injury. However, the SSI scores in the rats with the triple nerve crush injuries every week did not recover to normal range up to 8 weeks. Comparisons between the single and triple crush injuries every week were made with muscle fiber diameter and reinnervation of the tibialis anterior muscles. In the rats with the triple nerve crush injuries, the average diameters of 50 muscle fibers were significantly smaller than those with the single nerve crush injury at the point of 2, 3 and 4 weeks after the last crush injury. Reinnervation of the muscles was estimated by the ratio of synaptophysin positive nerve terminals in presynaptic membrane for -bungarotoxin positive neuroreceptors in postsynaptic membrane. At the point of 3 weeks after the last crush injury, the amount of reinnervation of the muscles in the rats with the triple nerve crush injuries was lower than that with the single nerve crush injury. In the rats with repeated sciatic nerve crush injuries at short intervals before recovering motor function, the function may not recover completely because of delay of the reinnervation and the atrophy of the muscles. doi:10.1016/j.neures.2010.07.1919 P3-q08 Neonatal EGF challenge permanently alters physiological property of dopaminergic neurons in the ventral tegmental area Hisaaki Namba , Hiroyuki Nawa Department of Mol. Neurobiol., Brain Res. Inst., Niigata University The neurotophic agent, epidermal growth factor (EGF) is implicated in dopamine-associated brain diseases. We found that neonatal exposure to EGF transiently enhances synaptic and neurochemical development of midbrain dopaminergic neurons, although we do not know how this postnatal EGF action influences later physiological property of this neuronal population. We prepared midbrain slice preparations from adult mice treated with EGF as neonates, and analyzed spontaneous firing activity of dopaminergic neurons and their sensitivity to a dopamine D2 receptor agonist. Cell-attached recording reveals that neonatal EGF treatment decreased the frequency of their pacemaker firing in the ventral tegmental area but not in the substantia nigra compacta. Subsequent whole-cell analysis indicates that EGF increased the threshold of membrane potential generating spikes. D2R agonist application to dopaminergic neurons attenuated their firing frequency in a dose dependent manner, but the magnitude of the suppression was not affected by EGF treatment. Thus, neurotrophic over-stimulation might perturb the physiological development of dopaminergic neurons and contribute to their pathophysiology of psychiatric diseases. doi:10.1016/j.neures.2010.07.1920 P3-q09 Gamma knife irradiation operates differentially on the neuronal subpopulations of the rat basal ganglia Nobuo Kouyama 1 , Yoko Katayama 1, Motohiro Hayashi 2, Yoriko Kawakami 1 1 Department Physiol, Tokyo Women’s Med Univ, Tokyo 2 Department Neurosurgery, Tokyo Women’s Med University, Tokyo To investigate the effect of gamma knife irradiation to the neuronal tissues, we have surveyed the histological disruption derived by the unilateral irradiation to the rat brain. Irradiation of high dose gamma ray provoked the necrosis of the brain tissues at the target area, and the rotation behaviors after prolonged survival. In this study, the effect of relatively weak, subnecrotic dose of irradiation and/or shorter survival period will be reported. After the irradiation, the nuclei of the basal ganglia between the striatum and the substantia nigra were systematically surveyed with immunohistochemistry. There was no necrotic lesion with this condition in macroscopic level. As we have reported previously, glial cells are susceptible to the irradiation. A low dose irradiation made the astrocytes to be swollen, giant cells in the target area. Microglia were found in activated form. The fate of the neurons depended on their subtypes. No obvious alteration of the immunoreactivity (IR) to calretinin (CR), tyrosine hydroxylase (TH), mu-opioid receptor (MOPR) was noticed. On the contrary, the IR to enkephalin (Enk) and calbindin (CB) were clearly enhanced over the irradiated hemisphere. They lost, however, the certain localization to the specific neuron subtypes and cytological structures, such as somata. The IR became diffused in the tissues, probably to the extracellular spaces, leaving the neuronal somata unlabeled like the negative stain. The patch/matrix segregation, which could be observed by the CB-IR in the unirradiated striatum, became unclear. Interestingly, however, it could be seen in the next sections by MOPR-IR. There are parvalbumin (PV)-IR neurons over the basal ganglia. The PV labeling of somata in the irradiated lateral globus pallidus, and of the fibers were reduced. These results indicate subtype specific operations of gamma knife in the nervous tissue. doi:10.1016/j.neures.2010.07.1921 P3-q10 Facilitating brain-machine interface research through data-sharing Satoshi Murata , Makoto Takemiya, Yukiyasu Kamitani