Indirect pathway between the primary auditory and visual cortices through layer V pyramidal neurons in V2L in mouse and the effects of bilateral enucleation.

Visual cortical areas are activated by auditory stimuli in blind mice. Direct heteromodal cortical connections have been shown between the primary auditory cortex (A1) and primary visual cortex (V1), and between A1 and secondary visual cortex (V2). Auditory afferents to V2 terminate in close proximity to neurons that project to V1, and potentially constitute an effective indirect pathway between A1 and V1. In this study, we injected a retrograde adenoviral vector that expresses enhanced green fluorescent protein under a synapsin promotor in V1 and biotinylated dextran amine as an anterograde tracer in A1 to determine: (i) whether A1 axon terminals establish synaptic contacts onto the lateral part of V2 (V2L) neurons that project to V1; and (ii) if this indirect cortical pathway is altered by a neonatal enucleation in mice. Complete dendritic arbors of layer V pyramidal neurons were reconstructed in 3D, and putative contacts between pre-synaptic auditory inputs and postsynaptic visual neurons were analysed using a laser-scanning confocal microscope. Putative synaptic contacts were classified as high-confidence and low-confidence contacts, and charted onto dendritic trees. As all reconstructed layer V pyramidal neurons received auditory inputs by these criteria, we conclude that V2L acts as an important relay between A1 and V1. Auditory inputs are preferentially located onto lower branch order dendrites in enucleated mice. Also, V2L neurons are subject to morphological reorganizations in both apical and basal dendrites after the loss of vision. The A1-V2L-V1 pathway could be involved in multisensory processing and contribute to the auditory activation of the occipital cortex in the blind rodent.

Neural connections between the lateral geniculate nucleus and visual cortex in vitro.

Neural connections were established in cocultures of rat visual cortex (VC) and lateral geniculate nucleus (LGN), which were isolated in early infancy. Morphological and electrophysiological studies showed that the cortical laminar organization of afferent and efferent connections in the coculture preparations was similar to that in the adult VC. The results indicate the existence of intrinsic mechanisms in VC and LGN that guide the formation of synaptic connections with the appropriate targets.

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.

Prenatal development of neural excitation in rat thalamocortical projections studied by optical recording.

To elucidate the formation of early thalamocortical synapses we recorded optical images with voltage-sensitive dyes from the cerebral cortex of prenatal rats by selective thalamic stimulation of thalamocortical slice preparations. At embryonic day (E) 17, thalamic stimulation elicited excitation that rapidly propagated through the internal capsule to the cortex. These responses lasted less than 15 ms, and were not affected by the application of glutamate receptor antagonists, suggesting that they might reflect presynaptic fiber responses. At E18, long-lasting (more than 300 ms) responses appeared in the internal capsule and in subplate. By E19, long-lasting responses increased in the cortical subplate. By E21, shortly before birth, the deep cortical layers were also activated in addition to the subplate. These long-lasting responses seen in the internal capsule and subplate were blocked by the antagonist perfusion, but the first spike-like responses still remained. The laminar location of the responses was confirmed in the same slices by Nissl staining and subplate cells were labeled by birthdating with bromodeoxyuridine at E13. Our results demonstrate that there is a few days delay between the arrival of thalamocortical axons at the subplate at E16 and the appearance of functional thalamocortical synaptic transmission at E19. Since thalamocortical connections are already functional within the subplate and in the deep cortical plate at embryonic ages, prenatal thalamocortical synaptic connections could influence cortical circuit formation before birth.

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 involvement 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.

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.

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.

Behavior of Japanese tree frogs under microgravity on MIR and in parabolic flight.

Japanese tree frogs (Hyla japonica) were flown to the space station MIR and spent eight days in orbit during December, 1990. Under microgravity, their postures and behaviors were observed and recorded. On the MIR, floating frogs stretched four legs out, bent their bodies backward and expanded their abdomens. Frogs on a surface often bent their neck backward and walked backwards. This behavior was observed on parabolic flights and resembles the retching behavior of sick frogs on land--a possible indicator of motion sickness. Observations on MIR were carried out twice to investigate the frog's adaptation to space. The frequency of failure in landing after a jump decreased in the second observation period. After the frogs returned to earth, readaptation processes were observed. The frogs behaved normally as early as 2.5 hours after landing.

Titanium casting: the surface reaction layer of castings obtained using ultra-low-temperature molds.

To examine whether the surface reaction layer of titanium castings can be reduced by lowering the mold temperature during casting, we cast titanium at three mold temperatures, including an ultra-low temperature produced by cooling the mold with liquid nitrogen, then measured the tensile strength and elongation of the castings. The titanium was cast using a centrifugal casting machine, and the molds were incinerated according to the manufacturers' instructions. Castings were then made with the molds at 200 degrees C, 600 degrees C, and an ultra-low temperature (-196 degrees C). The castability of titanium cast in the mold at the ultra-low temperature was good. The Vickers hardness near the surface layer of castings decreased as the mold temperature decreased.