Shun Tsuruno

Periodic Organization of a Major Subtype of Pyramidal Neurons in Neocortical Layer V

A major question in neocortical research is the extent to which neuronal organization is stereotyped. Previous studies have revealed functional clustering and neuronal interactions among cortical neurons located within tens of micrometers in the tangential orientation (orientation parallel to the pial surface). In the tangential orientation at this scale, however, it is unknown whether the distribution of neuronal subtypes is random or has any stereotypy. We found that the tangential arrangement of subcerebral projection neurons, which are a major pyramidal neuron subtype in mouse layer V, was not random but significantly periodic. This periodicity, which was observed in multiple cortical areas, had a typical wavelength of 30 μm. Under specific visual stimulation, neurons in single repeating units exhibited strongly correlated c-Fos expression. Therefore, subcerebral projection neurons have a periodic arrangement, and neuronal activity leading to c-Fos expression is similar among neurons in the same repeating units. These results suggest that the neocortex has a periodic functional micro-organization composed of a major neuronal subtype in layer V.

Persistent activation of protein kinase Cα is not necessary for expression of cerebellar long-term depression

Protein kinase Calpha (PKCalpha) plays a major role in the induction of long-term depression (LTD) in a cerebellar Purkinje cell (PC). The sequential activation model for classical PKC states that PKCalpha translocates to the plasma membrane by binding Ca(++) and then becomes fully activated by binding diacylglycerol (DAG), which enables estimation of the activity by monitoring its localization. Here, we performed simultaneous electrophysiological recording and fluorescence imaging in a cultured PC expressing GFP-tagged PKCalpha. When a PC was depolarized, PKCalpha transiently translocated to the plasma membrane in a Ca(++)-dependent manner. Application of membrane permeable DAG or the blocker of DAG lipase prolonged the translocation. These results suggest that the sequential activation model is applicable to PCs. Conjunctive applications of glutamate and depolarization pulse induced LTD, but did not prolong the translocation. Thus, our results imply that persistent activation of PKCalpha is not necessary for the expression of LTD.

Src-family protein tyrosine kinase negatively regulates cerebellar long-term depression

Protein phosphorylation is a major mechanism for the regulation of synaptic transmission. Previous studies have shown that several serine/threonine kinases are involved in the induction of long-term depression (LTD) at excitatory synapses on a Purkinje neuron (PN) in the cerebellum. Here, we show that Src-family protein tyrosine kinases (SFKs) are involved in the regulation of the LTD induction. Intracellular application of c-Src suppressed LTD. We also show that application of a SFK-selective inhibitor PP2 recovered LTD from the suppression caused by the inhibition of mGluR1 activity. These results indicate that SFKs negatively regulate the LTD induction at excitatory synapses on a cerebellar PN.

Lattice system of functionally distinct cell types in the neocortex

The basic modules of the neocortex The fundamental organization of excitatory and inhibitory neurons in the neocortex is still poorly understood. Subcerebral projection neurons, a major excitatory cell type in neocortical layer 5, form small cell clusters called microcolumns. Maruoka et al. examined large regions of mouse brain layer 5 and observed that thousands of these microcolumns make up a hexagonal lattice with a regular gridlike spacing. The other major layer 5 excitatory cell class, cortical projection neurons, also form microcolumns that interdigitate with those of the subcerebral projection neurons. Microcolumns received common presynaptic inputs and showed synchronized activity in many cortical areas. These microcolumns developed from nonsister neurons coupled by cell type–specific gap junctions, suggesting that their development is lineage-independent but guided by local electrical transmission. Science, this issue p. 610 Neocortical layer 5 is composed of microcolumns containing specific cell types that form a brainwide hexagonal lattice system. The mammalian neocortex contains many cell types, but whether they organize into repeated structures has been unclear. We discovered that major cell types in neocortical layer 5 form a lattice structure in many brain areas. Large-scale three-dimensional imaging revealed that distinct types of excitatory and inhibitory neurons form cell type–specific radial clusters termed microcolumns. Thousands of microcolumns, in turn, are patterned into a hexagonal mosaic tessellating diverse regions of the neocortex. Microcolumn neurons demonstrate synchronized in vivo activity and visual responses with similar orientation preference and ocular dominance. In early postnatal development, microcolumns are coupled by cell type–specific gap junctions and later serve as hubs for convergent synaptic inputs. Thus, layer 5 neurons organize into a brainwide modular system, providing a template for cortical processing.

Single-cell level multi-layered substructures of neocortical layer V

ber of extracellular axon guidance factors, their receptors and intracellular signaling molecules involved in controlling axon morphology, the signaling pathways in axon development remain to be elucidated. We have previously reported that R-Ras, a member of Ras family GTPases, plays an important role in axon specification and guidance. However, direct effectors of R-Ras in neurons have not been identified except PI3K. In this study, we report that lAfadin, an actin filament-binding protein having Ras association (RA) domain and PDZ domain, functions as an effector of R-Ras and regulates axon branching downstream of R-Ras in cultured hippocampal neurons. Pull-down and immunoprecipitaion assays showed that l-Afadin bound to active R-Ras. In Neuro-2a cells, constitutively active R-Ras recruited l-Afadin to the plasma membrane in a RA domain-dependent manner. In cultured neurons, overexpression of l-Afadin promoted axon branching, while knockdown of l-Afadin suppressed the branching activity. Overexpression of constitutively active R-Ras increased axon arborization and it was partially repressed by knockdown of l-Afadin. These results suggest that activated R-Ras induces axon branching in part by recruiting l-Afadin to the plasma membrane.

Precise three-dimensional functional micro-organization in neocortical layer V

Zic2 is a causal gene of holoprosencephaly (a dysgenesis of medial forebrain). Although it is broadly expressed in CNS, there was a difficulty to fully show its role due to its critical role in early embryogenesis. Here we developed a conditionally targeted Zic2 mutant mice and clarified its role in the development of dorsal cochlear nucleus (DCoN) and in the auditory function of mature mice. Soon after the neural tube closure, Zic2 and its close relatives (Zic1 and Zic3) are differentially expressed in hindbrain region along the rostrocaudal axis. Zic2 expression was dominant in the DCoN forming region. In Zic2 hypomorphic mutants (60% of wild type level), slight reduction of DCoN size was observed whereas the DCoN size reduction was severe in the midbrainhindbrain restricted Zic2 conditional knockout (CKO). Both granule cells and unipolar brush cells were decreased in DCoN. We observed the increased acoustic startle response and the altered auditory brain stem responses in both Zic2 hypomorphic and Zic2 CKO animals. Furthermore, we measured the activities of the primary auditory cortices during various sound stimuli application by means of autofluorescence imaging. These results indicated that optimal Zic2 gene dosage is a critical parameter for the auditory neural circuit formation and the auditory function. Further analyses using the Zic2 mutants would be beneficial for understanding physiological regulation of auditory information processing in mammalian. Research fund: RIKEN BSI funds.

Analysis of the formation of single-cell level micro-organization in neocortical layer V

We have previously found that layer V projection neurons that have different axonal projection or gene expression make precise three-dimensional organization in the radial orientation, projection neurons form thin sublayers with single-cell precision, and multiple sublayers are stacked in layer V. As a first step to investigate mechanisms of the multi-layer formation, we analyzed correlations between axonal projections and gene expression. The result showed that the sublayers defined by gene expression precisely correlate with the sublayers defined by axonal projection targets. Our analysis suggest that layer V is precisely subdivided into multiple sublayers of functionally different neuronal types and that microcircuits involving layer V projection neurons may be strictly specified by molecular mechanisms. Analysis of over-expression experiments will be discussed.

Micro cortical unit: A functional unit of neurons periodically repeated in neocortical layer V

Understanding how sensory representations are transformed at each brain region is a fundamental step towards obtaining the entire picture of sensory processing. To accomplish this, one would like to examine the relationship between stimulus evoked spike trains of input and output neurons in vivo. To further understand the mechanisms underlying this transformation, one would like to investigate the properties of synapses as well as the pattern of connectivity between populations of input and output neurons. Here we used the Drosophila antennal lobe, an analogue of the vertebrate olfactory bulb, as a model system to achieve these goals. We characterized the synapse between olfactory receptor neurons (ORNs) and projection neurons (PNs) and found that this synapse is unusually strong, reliable and exhibits prominent shortterm depression. These synaptic properties help explain how odor-evoked spikes are transformed in the antennal lobe in vivo. Further, we investigated the pattern of connectivity between ORNs and PNs. In flies, as in vertebrates, a group of ORNs expressing the same olfactory receptor converge to the same compartment called glomerulus. Whether each second order neuron receives input from all or a subset of ORNs was unknown in any species. Using dual recordings from PNs in the same glomerulus (homotypic PNs), we found that every PN listens to all the ORNs with in the glomerulus. Because ORNs function independently, pooling of ORN inputs will increase the signal to noise ratio of the total input to PNs. Thus, convergence of ORNs helps achieve the high sensitivity of PNs to odors. Moreover, this all-to-all connectivity produces correlated spikes between homotypic PNs, which may have implications on the downstream processing. In sum, these advances demonstrate that in vivo physiology is feasible in the Drosophila brain, and should permit a new integration of genetic, synaptic, cellular and systems approaches to investigating neural processing.