Hidenobu Mizuno

Evidence for Activity-Dependent Cortical Wiring: Formation of Interhemispheric Connections in Neonatal Mouse Visual Cortex Requires Projection Neuron Activity

Neuronal activity plays a pivotal role in shaping neuronal wiring. We investigated the role of neuronal activity in the formation of interhemispheric (callosal) axon projections in neonatal mouse visual cortex. Axonal labeling with enhanced green fluorescent protein (GFP) was used to demonstrate spatially organized pattern of callosal projections: GFP-labeled callosal axons from one hemisphere projected densely to a narrowly restricted region at the border between areas 17 and 18 in the contralateral hemisphere, in which they terminated in layers 1–3 and 5. This region- and layer-specific innervation pattern developed by postnatal day 15 (P15). To explore the role of neuronal activity of presynaptic and postsynaptic neurons in callosal connection development, an inwardly rectifying potassium channel, Kir2.1, was expressed in callosal projection neurons and their target postsynaptic neurons. Kir2.1 overexpression reduced the firing rate of cortical neurons. Kir2.1 overexpression in callosal projection neurons disturbed the growth of axons and their arbors that normally occurs between P7 and P13, whereas that in postsynaptic neurons had limited effect on the pattern of presynaptic callosal axon innervation. In addition, exogenous expression of a gain-of-function Kir2.1 mutant channel found in patients with a familial heart disease caused severe deficits in callosal axon projections. These results suggest that projection neuron activity plays a crucial role in interhemispheric connection development and that enhanced Kir2.1 activity can affect cortical wiring.

Control of Cortical Axon Elongation by a GABA-Driven Ca2+/Calmodulin-Dependent Protein Kinase Cascade

Ca2+ signaling plays important roles during both axonal and dendritic growth. Yet whether and how Ca2+ rises may trigger and contribute to the development of long-range cortical connections remains mostly unknown. Here, we demonstrate that two separate limbs of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)–CaMKI cascades, CaMKK–CaMKIα and CaMKK–CaMKIγ, critically coordinate axonal and dendritic morphogenesis of cortical neurons, respectively. The axon-specific morphological phenotype required a diffuse cytoplasmic localization and a strikingly α-isoform-specific kinase activity of CaMKI. Unexpectedly, treatment with muscimol, a GABAA receptor agonist, selectively stimulated elongation of axons but not of dendrites, and the CaMKK–CaMKIα cascade critically mediated this axonogenic effect. Consistent with these findings, during early brain development, in vivo knockdown of CaMKIα significantly impaired the terminal axonal extension and thereby perturbed the refinement of the interhemispheric callosal projections into the contralateral cortices. Our findings thus indicate a novel role for the GABA-driven CaMKK–CaMKIα cascade as a mechanism critical for accurate cortical axon pathfinding, an essential process that may contribute to fine-tuning the formation of interhemispheric connectivity during the perinatal development of the CNS.

Pre-synaptic and post-synaptic neuronal activity supports the axon development of callosal projection neurons during different post-natal periods in the mouse cerebral cortex

Callosal projection neurons, one of the major types of projection neurons in the mammalian cerebral cortex, require neuronal activity for their axonal projections [H. Mizuno et al. (2007) J. Neurosci., 27, 6760–6770; C. L. Wang et al. (2007) J. Neurosci., 27, 11334–11342]. Here we established a method to label a few callosal axons with enhanced green fluorescent protein in the mouse cerebral cortex and examined the effect of pre‐synaptic/post‐synaptic neuron silencing on the morphology of individual callosal axons. Pre‐synaptic/post‐synaptic neurons were electrically silenced by Kir2.1 potassium channel overexpression. Single axon tracing showed that, after reaching the cortical innervation area, green fluorescent protein‐labeled callosal axons underwent successive developmental stages: axon growth, branching, layer‐specific targeting and arbor formation between post‐natal day (P)5 and P9, and the subsequent elaboration of axon arbors between P9 and P15. Reducing pre‐synaptic neuronal activity disturbed axon growth and branching before P9, as well as arbor elaboration afterwards. In contrast, silencing post‐synaptic neurons disturbed axon arbor elaboration between P9 and P15. Thus, pre‐synaptic neuron silencing affected significantly earlier stages of callosal projection neuron axon development than post‐synaptic neuron silencing. Silencing both pre‐synaptic and post‐synaptic neurons impaired callosal axon projections, suggesting that certain levels of firing activity in pre‐synaptic and post‐synaptic neurons are required for callosal axon development. Our findings provide in‐vivo evidence that pre‐synaptic and post‐synaptic neuronal activities play critical, and presumably differential, roles in axon growth, branching, arbor formation and elaboration during cortical axon development.

Transcallosal Projections Require Glycoprotein M6-Dependent Neurite Growth and Guidance

The function of mature neurons critically relies on the developmental outgrowth and projection of their cellular processes. It has long been postulated that the neuronal glycoproteins M6a and M6b are involved in axon growth because these four-transmembrane domain-proteins of the proteolipid protein family are highly enriched on growth cones, but in vivo evidence has been lacking. Here, we report that the function of M6 proteins is required for normal axonal extension and guidance in vivo. In mice lacking both M6a and M6b, a severe hypoplasia of axon tracts was manifested. Most strikingly, the corpus callosum was reduced in thickness despite normal densities of cortical projection neurons. In single neuron tracing, many axons appeared shorter and disorganized in the double-mutant cortex, and some of them were even misdirected laterally toward the subcortex. Probst bundles were not observed. Upon culturing, double-mutant cortical and cerebellar neurons displayed impaired neurite outgrowth, indicating a cell-intrinsic function of M6 proteins. A rescue experiment showed that the intracellular loop of M6a is essential for the support of neurite extension. We propose that M6 proteins are required for proper extension and guidance of callosal axons that follow one of the most complex trajectories in the mammalian nervous system.

Activity-dependent development of interhemispheric connections in the visual cortex.

Interhemispheric axon fibers connect the two cerebral cortical hemispheres via the corpus callosum and function to integrate information between the hemispheres. In the development of callosal connections, an early phase involves axon guidance molecules and a later phase requires neuronal activity. In addition to the well-studied role of sensory-driven neuronal activity, recent studies have demonstrated an essential role of callosal neuron firing activity in forming axonal projections and dendritic maturation during the developmental period before sensory input is available. Results suggest that factors affecting the cellular excitability of developing callosal neurons can influence the establishment of interhemispheric connections. Possible synaptic and non-synaptic mechanisms for activity-dependent axonal projections are discussed.

Developmental RacGAP α2-Chimaerin Signaling Is a Determinant of the Morphological Features of Dendritic Spines in Adulthood

Morphological characteristics of dendritic spines form the basis of cognitive ability. However, molecular mechanisms involved in fine-tuning of spine morphology during development are not fully understood. Moreover, it is unclear whether, and to what extent, these developmental mechanisms determine the normal adult spine morphological features. Here, we provide evidence that α2-isoform of Rac-specific GTPase-activating protein α-chimaerin (α2-chimaerin) is involved in spine morphological refinement during late postnatal period, and furthermore show that this developmental α2-chimaerin function affects adult spine morphologies. We used a series of mice with global and conditional knock-out of α-chimaerin isoforms (α1-chimaerin and α2-chimaerin). α2-Chimaerin disruption, but not α1-chimaerin disruption, in the mouse results in an increased size (and density) of spines in the hippocampus. In contrast, overexpression of α2-chimaerin in developing hippocampal neurons induces a decrease of spine size. Disruption of α2-chimaerin suppressed EphA-mediated spine morphogenesis in cultured developing hippocampal neurons. α2-Chimaerin disruption that begins during the juvenile stage results in an increased size of spines in the hippocampus. Meanwhile, spine morphologies are unaltered when α2-chimaerin is deleted only in adulthood. Consistent with these spine morphological results, disruption of α2-chimaerin beginning in the juvenile stage led to an increase in contextual fear learning in adulthood; whereas contextual learning was recently shown to be unaffected when α2-chimaerin was deleted only in adulthood. Together, these results suggest that α2-chimaerin signaling in developmental stages contributes to determination of the morphological features of adult spines and establishment of normal cognitive ability. SIGNIFICANCE STATEMENT Recent studies of neurodevelopmental disorders in humans and their animal models have led to an attractive hypothesis that spine morphogenesis during development forms the basis of adult cognition. In particular, the roles of Rac and its regulators, such as Rac-specific GTPase-activating proteins (RacGAPs) and Rac guanine nucleotide exchange factors, are a topic of focus in spine morphogenesis and cognitive ability. Using a series of mice with global and conditional knock-out (KO) of RacGAP α-chimaerin isoforms (α1-chimaerin and α2-chimaerin), we provide compelling evidence demonstrating that α2-chimaerin is involved in spine morphological refinement during late postnatal development and that this developmental α2-chimaerin function affects adult spine morphologies. Furthermore, our results clearly showed that α2-chimaerin signaling during late postnatal development contributes to normal cognitive ability in adult mice.

Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo.

Here we describe “Supernova” series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

High intensity single cell labeling reveals precise developmental processes of barrel neuron dendrites in somatosensory cortex of neonatal mice

P2-f14 Involvement of Olig2 in the neural circuit formation in the fetal mouse forebrain Katsuhiko Ono 1,5 , Carlos M. Parras 2, Hirohide Takebayashi 3, Kenji Shimamura 4, Hitoshi Gotoh 1,5, Kazuhiro Ikenaka 5 1 Dept. of Biol., Kyoto Pref. Univ. Medicine 2 Institute of the Brain and Spinal Cord (ICM), Inserm-UPMC, Paris, France 3 Dept. of Morphological Neural Science, Grad. Sch. of Medical Sciences, Kumamoto Univ., Kumamoto, Japan 4 Dept. of Brain Morphogenesis, Inst. Mol. Embryol. Genetics, Kumamoto Univ., Kumamoto, Japan 5 Div. of Mol. Neurobiol. Bioinfo., NIPS, Okazaki, Japan

Roles of pre- and post-synaptic neuron activity in interhemispheric connection development in the mouse visual cortex

Chondroitin sulfate (CS) chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins, such as growth factors. Our previous study suggested that CS-D type structure generated by UST is important for the development of Purkinje cells. In this study, we analyzed the expression patterns of several CS sulfotransferases (UST, GalNAc46ST, D4ST) in the postnatal mouse cerebellum. While the expression of UST gene increased from P1 to P14, that of GalNAc4S-6ST and D4ST genes decreased during this period. Using in situ hybridization, it was revealed that these genes were expressed by Purkinje cells, Bergmann glia, and granular cells. These results suggest that the structural variation of CS chains may regulate Purkinje cell-Bergmann glia interaction and granule cell migration.