Tohru Kurotani

Laminar specificity of extrinsic cortical connections studied in coculture preparations

The formation of specific neural connections in the cerebral cortex was studied using organotypic coculture preparations composed of subcortical and cortical regions. Morphological and electrophysiological analysis indicated that several cortical efferent and afferent connections, such as the corticothalamic, thalamocortical, corticocortical, and corticotectal connections, were established in the cocultures with essentially the same laminar specificity as that found in the adult cerebral cortex, but without specificity of sensory modality. This suggests the existence of a cell-cell recognition system between cortical or subcortical neurons and their final targets. This interaction produces lamina-specific connections, but is probably insufficient for the formation of the modality-specific connections.

Functional Thalamocortical Synapse Reorganization from Subplate to Layer IV during Postnatal Development in the Reeler-Like Mutant Rat (Shaking Rat Kawasaki)

Transient synapse formation between thalamic axons and subplate neurons is thought to be important in thalamocortical targeting. Shaking rat Kawasaki (SRK), having reversed cortical layering similarly observed in reeler mouse, provides an interesting model system to test this idea. The spatial and temporal pattern of excitation was investigated using optical recording with voltage-sensitive dyes in thalamocortical slice preparations from SRK. At postnatal day 0 (P0), a strong optical response was elicited within the superplate of the SRK in the cell layer corresponding to subplate in wild-type (WT) rats. By P3, this response rapidly descended into deep cortical layers comprised of layer IV cells, as identified with 5-bromo-2′-deoxyuridine birthdating at embryonic day 17. During the first 3 postnatal days, both the subplate and cortical plate responses were present, but by P7, the subplate response was abolished. Tracing individual axons in SRK revealed that at P0-P3, a large number of thalamocortical axons reach the superplate, and by P7-P10, the ascending axons develop side branches into the lower or middle cortical layers. Synaptic currents were also demonstrated in WT subplate cells and in SRK superficial cortical cells using whole-cell recording. These currents were elicited monosynaptically, because partial AMPA current blockade did not modify the latencies. These results suggest that the general developmental pattern of synapse formation between thalamic axons and subplate (superplate) neurons in WT and SRK is very similar, and individual thalamic arbors in cortex are considerably remodeled during early postnatal development to find layer IV equivalent neurons.

Development of functional thalamocortical synapses studied with current source-density analysis in whole forebrain slices in the rat.

We analysed the laminar distribution of transmembrane currents from embryonic (E) day 17 until adulthood after selective thalamic stimulation in slices of rat forebrain to study the development of functional thalamocortical and cortico-cortical connections. At E18 to birth a short-latency current sink was observed in the subplate and layer 6, which was decreased, but not fully abolished in a cobalt containing solution or after the application of glutamate receptor blockers (APV and DNQX). This indicated that embryonic thalamic axons were capable of conducting action potentials to the cortex and some of them had already formed functional synapses there. Between birth and P3, when thalamic axons were completing their upward growth, a sink gradually appeared more superficially in the dense cortical plate and synchronously, a current source aroused in layer 5. Both sinks and sources completely disappeared after blocking synaptic transmission. The adult-like distribution of CSDs became apparent after P7. The component in layer 6 cannot be blocked completely after this age suggesting antidromic activation. This study demonstrated that cells of the lowest layers of the cortex received functional thalamic input before birth and that thalamocortical axons formed synapses with more superficial cells as they grew into the cortical plate.

Dynamic Expression of Cadherins Regulates Vocal Development in a Songbird

Background Since, similarly to humans, songbirds learn their vocalization through imitation during their juvenile stage, they have often been used as model animals to study the mechanisms of human verbal learning. Numerous anatomical and physiological studies have suggested that songbirds have a neural network called ‘song system’ specialized for vocal learning and production in their brain. However, it still remains unknown what molecular mechanisms regulate their vocal development. It has been suggested that type-II cadherins are involved in synapse formation and function. Previously, we found that type-II cadherin expressions are switched in the robust nucleus of arcopallium from cadherin-7-positive to cadherin-6B-positive during the phase from sensory to sensorimotor learning stage in a songbird, the Bengalese finch. Furthermore, in vitro analysis using cultured rat hippocampal neurons revealed that cadherin-6B enhanced and cadherin-7 suppressed the frequency of miniature excitatory postsynaptic currents via regulating dendritic spine morphology. Methodology/Principal Findings To explore the role of cadherins in vocal development, we performed an in vivo behavioral analysis of cadherin function with lentiviral vectors. Overexpression of cadherin-7 in the juvenile and the adult stages resulted in severe defects in vocal production. In both cases, harmonic sounds typically seen in the adult Bengalese finch songs were particularly affected. Conclusions/Significance Our results suggest that cadherins control vocal production, particularly harmonic sounds, probably by modulating neuronal morphology of the RA nucleus. It appears that the switching of cadherin expressions from sensory to sensorimotor learning stage enhances vocal production ability to make various types of vocalization that is essential for sensorimotor learning in a trial and error manner.

Neurotrophin-3 Is Involved in the Formation of Apical Dendritic Bundles in Cortical Layer 2 of the Rat

Apical dendritic bundles from pyramidal neurons are a prominent feature of cortical neuropil but with significant area specializations. Here, we investigate mechanisms of bundle formation, focusing on layer (L) 2 bundles in rat granular retrosplenial cortex (GRS), a limbic area implicated in spatial memory. By using microarrays, we first searched for genes highly and specifically expressed in GRS L2 at postnatal day (P) 3 versus GRS L2 at P12 (respectively, before and after bundle formation), versus GRS L5 (at P3), and versus L2 in barrel field cortex (BF) (at P3). Several genes, including neurotrophin-3 (NT-3), were identified as transiently and specifically expressed in GRS L2. Three of these were cloned and confirmed by in situ hybridization. To test that NT-3–mediated events are causally involved in bundle formation, we used in utero electroporation to overexpress NT-3 in other cortical areas. This produced prominent bundles of dendrites originating from L2 neurons in BF, where L2 bundles are normally absent. Intracellular biocytin fills, after physiological recording in vitro, revealed increased dendritic branching in L1 of BF. The controlled ectopic induction of dendritic bundles identifies a new role for NT-3 and a new in vivo model for investigating dendritic bundles and their formation.

Altered spatial patterns of functional thalamocortical connections in the barrel cortex after neonatal infraorbital nerve cut revealed by optical recording

In rodents, the somatosensory cortex has a cell aggregation cluster termed the barrel, reflecting a whisker vibrissa, and this barrel formation is disrupted by infraorbital nerve cut at birth. In the present study, we prepared thalamocortical slice preparations from rats that received infraorbital nerve cut either at birth or at postnatal day (P) 7 and those from normal rats, recorded the optical response reflecting neural excitation in the somatosensory cortex with a voltage-sensitive dye (RH482) and compared the optical responses from lesioned rats with those from normal rats. In normal rats at P10, the optical response elicited electrically by thalamic stimulation propagated to the cortex, and then several patchy clusters appeared in layer IV. The size and location of these patchy responses precisely matched either barrels identified by cytochrome oxidase staining or terminal arbors of thalamocortial axons stained with biotinylated dextran amine. In contrast, at P10 in P0-lesioned rats, clusters having a wider horizontal width but smaller amplitude than those seen in normal rats appeared in layer IV. Correspondingly, neither cytochrome oxidase staining nor biotinylated dextran amine labeling of thalamocortical axons showed any barrel-like clusters or glomerular axon terminals. Likewise, at P5-P6, the tangential width of clusters in layer IV were larger than that in normal rats. At P10 in P7-lesioned rats, small cluster-matched barrels were seen in the optical response as well as in normal rats. These results suggest that P0 infraorbital nerve cut interrupted segregation of functional synapses into the barrels and retarded the maturation of thalamocortical transmission.

Protein and RNA synthesis-dependent and -independent LTPs in developing rat visual cortex

MULTIPLE forms of synaptic potentiation have been described, but their invovement in development versus learning is unknown. To address this, we examined whether long-term potentiation (LTP) in visual cortex requires protein or RNA synthesis using slice preparations. Theta-burst stimulation of white matter induced two distinct types of LTP in layer 4. A slowly developing LTP, preferentially induced in juveniles, was blocked by protein and RNA synthesis inhibitors and was L-type calcium channel dependent. A quickly developing LTP, induced in juveniles and adults, was independent of macromolecular synthesis and required N-methyl-D-aspartate receptor activation. Thus, slow LTP might account for developmental plasticity in visual cortex including the activity-dependent refinement of neural circuitry while fast LTP might underlie the changes in synaptic strength that may participate in visual learning and memory.

Development of neural connections between visual cortex and transplanted lateral geniculate nucleus in rats.

The development of neural connections between transplanted lateral geniculate nucleus (LGN) and host visual cortex (VC) was studied in slice preparations obtained from rat brain in which a fetal (embryonic day 15-17) rat LGN was transplanted to the white matter underlying the VC of a neonate rat (postnatal day 0-1). Placing a fluorescent dye (DiI) in the transplant of the fixed slices revealed that retrogradely labeled cortical cells projecting to the transplant were broadly distributed through layers II to VI at 1 week after transplantation. Three weeks after transplantation, these cells were virtually confined to layer VI. Likewise, anterograde labeling showed that cells in the transplant sent axons up to layer I with a few branches at 1 week after transplantation, while the axons were found to terminate at layer IV with many arborizations at 3 weeks after transplantation. These observations were supported by electrophysiological studies. Analysis of the antidromic responses of the cortical cells to stimulation of the transplant showed that the efferent cells projecting to the transplant were broadly distributed in layers II-VI at 1 week after transplantation, while they were virtually restricted to layer VI at 3 weeks after transplantation. Current source-density analysis of the field potentials and intracellular analysis of the synaptic potentials in the cortical cells demonstrated that geniculocortical connections were broadly established in layers II-VI at 1 week after transplantation, and were localized to layer IV and VI at 3 weeks after transplantation. These results suggest that the development of neural connections between transplanted LGN and host VC is characterized by an initial broad distribution of afferent and efferent connections without laminar specificity, and by later selection of appropriate connections to yield lamina-specific connections comparable to those in normal adult VC.

In vitro approach to visual cortical development and plasticity

The neural circuitry in the visual cortex is characterized by two basic types of organization. One is a laminar organization determining the extrinsic and intrinsic neural connections of cortical cells according to their cortical depth, and the other is a columnar organization where cortical cells are arranged perpendicularly according to their response selectivities. It is known that the columnar organization comprises the postnatal structures dependent on the visual experience, while the laminar organization comprises the prenatal structures unmodified by visual experience. We have investigated the interplay between the pre- and postnatal mechanisms using various in vitro preparations, including visual cortical slices, and transplant and co-culture preparations. It was shown in lateral geniculate and visual cortex transplants and co-cultures including the visual cortex lateral geniculate nucleus that all laminar structures are expressed in these preparations according to the prenatal mechanisms. It was also shown in slice preparations that the details of these circuitries are plastic and modifiable by the visual input, although their basic framework is determined prenatally.

Pyramidal neurons in the superficial layers of rat retrosplenial cortex exhibit a late-spiking firing property

The rodent granular retrosplenial cortex (GRS) is reciprocally connected with the hippocampus. It is part of several networks implicated in spatial learning and memory, and is known to contain head-direction cells. There are, however, few specifics concerning the mechanisms and microcircuitry underlying its involvement in spatial and mnemonic functions. In this report, we set out to characterize intrinsic properties of a distinctive population of small pyramidal neurons in layer 2 of rat GRS. These neurons, as well as those in adjoining layer 3, were found to exhibit a late-spiking (LS) firing property. We established by multiple criteria that the LS property is a consequence of delayed rectifier and A-type potassium channels. These were identified as Kv1.1, Kv1.4 and Kv4.3 by Genechip analysis, in situ hybridization, single-cell reverse transcriptase-polymerase chain reaction, and pharmacological blockade. The LS property might facilitate comparison or integration of synaptic inputs during an interval delay, consistent with the proposed role of the GRS in memory-related processes.