Yoshiaki Tagawa

Specific deficit of the ON response in visual transmission by targeted disruption of the mGIuR6 gene

Taking advantage of the restricted expression of metabotropic glutamate receptor subtype 6 (mGluR6) in retinal ON bipolar cells, we generated knockout mice lacking mGluR6 expression. The homozygous mutant mice showed a loss of ON responses but unchanged OFF responses to light. The mutant mice displayed no obvious changes in retinal cell organization nor in the projection of optic fibers to the brain. Furthermore, the mGluR6-deficient mice showed visual behavioral responses to light stimulation as examined by shuttle box avoidance behavior experiments using light exposure as a conditioned stimulus. The results demonstrate that mGluR6 is essential in synaptic transmission to the ON bipolar cell and that the OFF response provides an important means for transmitting visual information.

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.

The Whole Nucleotide Sequence and Chromosomal Localization of the Gene for Human Metabotropic Glutamate Receptor Subtype 6

Metabotropic glutamate receptor subtype 6 (mGluR6) is restrictedly expressed in the retinal ON bipolar cells and ablation of mouse mGluR6 by gene targeting results in a loss of ON responses to light stimulus and impairs the detection of visual contrasts. We have isolated genomic clones containing the human mGluR6 gene and determined the whole nucleotide sequence of the mGluR6 gene. The transcription initiation site of the human mGluR6 gene has been identified using primer extension analysis in combination with reverse transcriptase‐mediated polymerase chain reaction analysis of human retinal RNA, while the termination of the mGluR6 mRNA has been assigned by the analysis of rapid amplification of 3′‐cDNA ends. The human mGluR6 gene consists of 16 742 base pairs with 10 exons separated by nine introns. The human mGluR6 is composed of 877 amino acid residues with a signal peptide of 24 amino acid residues and the mature protein shows a 94.6% homology with the rat counterpart. A CpG‐rich island is present at exon 1 and its preceding putative promoter region and this unusual sequence, like several tissue‐specific genes, may be important for a specific expression of the mGluR6 gene in the retinal bipolar cells. The human mGluR6 gene has been mapped to chromosome 5q35 by the analyses of blot hybridization of a DNA panel of human/mouse/hamster somatic cell hybrids and fluorescence in situ hybridization of human chromosomes. This study should provide the genetic basis for not only better understanding the molecular mechanism underlying a tissue‐specific expression of the mGluR6 gene but also exploring a potential defect in human mGluR6 in a certain inherited eye disease.

Spatio‐temporal control of neural activity in vivo using fluorescence microendoscopy

Controlling neural activity with high spatio‐temporal resolution is desired for studying how neural circuit dynamics control animal behavior. Conventional methods for manipulating neural activity, such as electrical microstimulation or pharmacological blockade, have poor spatial and/or temporal resolution. Algal protein channelrhodopsin‐2 (ChR2) enables millisecond‐precision control of neural activity. However, a photostimulation method for high spatial resolution mapping in vivo is yet to be established. Here, we report a novel optical/electrical probe, consisting of optical fiber bundles and metal electrodes. Optical fiber bundles were used as a brain‐insertable endoscope for image transfer and stimulating light delivery. Light‐induced activity from ChR2‐expressing neurons was detected with electrodes bundled to the endoscope, enabling verification of light‐evoked action potentials. Photostimulation through optical fiber bundles of transgenic mice expressing ChR2 in layer 5 cortical neurons resulted in single‐whisker movement, indicating spatially restricted activation of neurons in vivo. The probe system described here and a combination of various photoactive molecules will facilitate studies on the causal link between specific neural activity patterns and behavior.

Control of Spontaneous Ca2+ Transients Is Critical for Neuronal Maturation in the Developing Neocortex

Neural activity plays roles in the later stages of development of cortical excitatory neurons, including dendritic and axonal arborization, remodeling, and synaptogenesis. However, its role in earlier stages, such as migration and dendritogenesis, is less clear. Here we investigated roles of neural activity in the maturation of cortical neurons, using calcium imaging and expression of prokaryotic voltage-gated sodium channel, NaChBac. Calcium imaging experiments showed that postmigratory neurons in layer II/III exhibited more frequent spontaneous calcium transients than migrating neurons. To test whether such an increase of neural activity may promote neuronal maturation, we elevated the activity of migrating neurons by NaChBac expression. Elevation of neural activity impeded migration, and induced premature branching of the leading process before neurons arrived at layer II/III. Many NaChBac-expressing neurons in deep cortical layers were not attached to radial glial fibers, suggesting that these neurons had stopped migration. Morphological and immunohistochemical analyses suggested that branched leading processes of NaChBac-expressing neurons differentiated into dendrites. Our results suggest that developmental control of spontaneous calcium transients is critical for maturation of cortical excitatory neurons in vivo: keeping cellular excitability low is important for migration, and increasing spontaneous neural activity may stop migration and promote dendrite formation.

Dysfunction of KCNK Potassium Channels Impairs Neuronal Migration in the Developing Mouse Cerebral Cortex

Development of the cerebral cortex depends partly on neural activity, but the identity of the ion channels that might contribute to the activity-dependent cortical development is unknown. KCNK channels are critical determinants of neuronal excitability in the mature cerebral cortex, and a member of the KCNK family, KCNK9, is responsible for a maternally transmitted mental retardation syndrome. Here, we have investigated the roles of KCNK family potassium channels in cortical development. Knockdown of KCNK2, 9, or 10 by RNAi using in utero electroporation impaired the migration of late-born cortical excitatory neurons destined to become Layer II/III neurons. The migration defect caused by KCNK9 knockdown was rescued by coexpression of RNAi-resistant functional KCNK9 mutant. Furthermore, expression of dominant-negative mutant KCNK9, responsible for the disease, and electrophysiological experiments demonstrated that ion channel function was involved in the migration defect. Calcium imaging revealed that KCNK9 knockdown or expression of dominant-negative mutant KCNK9 increased the fraction of neurons showing calcium transients and the frequency of spontaneous calcium transients. Mislocated neurons seen after KCNK9 knockdown stayed in the deep cortical layers, showing delayed morphological maturation. Taken together, our results suggest that dysfunction of KCNK9 causes a migration defect in the cortex via an activity-dependent mechanism.

Neuronal activity is not required for the initial formation and maturation of visual selectivity

Neuronal activity is important for the functional refinement of neuronal circuits in the early visual system. At the level of the cerebral cortex, however, it is still unknown whether the formation of fundamental functions such as orientation selectivity depends on neuronal activity, as it has been difficult to suppress activity throughout development. Using genetic silencing of cortical activity starting before the formation of orientation selectivity, we found that the orientation selectivity of neurons in the mouse visual cortex formed and matured normally despite a strong suppression of both spontaneous and visually evoked activity throughout development. After the orientation selectivity formed, the distribution of the preferred orientations of neurons was reorganized. We found that this process required spontaneous activity, but not visually evoked activity. Thus, the initial formation and maturation of orientation selectivity is largely independent of neuronal activity, and the initial selectivity is subsequently modified depending on neuronal activity.

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.