Visualization of axonal guidance and patterning in transgenic mice expressing green fluorescent protein

S297 VISUALIZATION IN TRANSGENIC

669

OF AXONAL GUIDANCE AND PATTERNING MICE EXPRESSING GREEN FLUORESCENT

PROTEIN

YUKI HATTORI, MICHIKO SAITO, NATSUKI MATSUSHITA, KAZUTO KOBAYASHI Animal Molecular Technology, Research and Education Center for Genetic Information, Nara Institute of Science and Technology,

8916-5, Takayama,

Ikoma, Nara 630-0101

To study development of accurate neuronal connections in the mammalian CNS. it is necessary system for monitoring visualizing

catecholaminergic

control of tyrosine expression

axonal guidance neurons

hydroxylase

and projection

with green fluorescent

or dopamine

P-hydroxylase

of the gene. Also, tau-GFP, axon-targeted

brains, the GFP fluorescence

of specific neurons.

brain development

These transgenic

using tissue culture or whole-mounted

REMOVAL ECTOPIC

670

neurons.

protein (GFP). gene promoter

to have an experimantal

transgenic

mouse lines for

In these mice, GFP was expressed which drives catecholaminergic

under the

neuron-specific

fusion protein was used as an imaging marker. In the adult transgenic

was detected in various regions,

where TH immunoreactivity was colocalized. The fluorescence terminals of catecholaminergic

We have generated

such as hypothalamus, was observed

substantia nigra, and locus coeruleus,

in the cell bodies, axon bundles,

and synaptic

lines are useful for tracing cell lineage and axonal guidance during brain culture.

OF

PSA CAIJSEES ABERRABT MOSSY FIBER SYBAPTOGEBBSIS 119 THE HIPPOCAMPUS

INRERVATIOI

ABD

TATSUNORI SEKI"', URS RUTISHAUSER', YASUMASA ARA12 ‘Dept. ‘Depts.

of

of

Anatomy, Genetics,

Juntendo Univ. Case Western

Sch. of Medicine, Reserve University,

Bunkyo-ku, Cleveland,

Tokyo 113-8421, OH 44106

The mossy fiber axons of both the developing and adult dentate gyrus express the highly polysialylated form of NCAM as they innervate the dendritic processes of pyramidal cells in the CA3 region of the hippocampus. The present study uses PSAdeficient and NCAM mutant mice to evaluate the role of PSA in mossy fiber development. The results indicate that removal of PSA by either specific enzymatic degradation or mutation of the NCAM-110 isoform that carries PSA in the brain, causes an aberrant and persistent innervation of the pyramidal cell layer by mossy fibers, including excessive collateral sprouting and/or defasciculation of these processes as well as formation of ectopic mossy fiber synaptic boutons. These results are considered in terms of two possible effects of PSA removal: an increase in the number of mossy fibers that can grow into the pyramidal cell layer, and an inhibition of process retraction by formation of stable junctions including synapses.

671 HIROSHI

IN V7TRO RECONSTRUCTION COCULTURE TAKUMA,

Dept. of Neurology.

MASAKI SAKURAI Div. of Neuroscience.,

OF THE CORTICOSPINAL

PROJECTION

IN ORGANOTYPIC

SLICE

and ICHIRO KANAZAWA Graduate Sch. of Med. Univ. of Tokyo, Tokyo 113-8155: Japan

The slices of the cerebral cortex and the spinal cord were prepared from newborn rats and cocultured on the pored membrane for 16-24 days. Neural connections were studied morphologically with biocytin (anterogradely), horseradish peroxidase and DiI (retrogradely). Substantial neural connections were formed between the cortex and spinal cord slices. Cortical neurons extended axon to the spinal cord were located mainly in the deeper layer of the cortex. In electrophysiological study, a stimulating electrode was placed in the deeper layer of the cortical explant. Extracellular or intracellular recordings were made from the gray matter of the spinal explant with glass microelectrode. When the deeper layer of the cortex was stimulated, the tield potential consisted of early sharp, presynaptic volley and late slow component, postsynaptic response was recorded from the spinal cord. The amplitude of the field potentials were examined among the four coculture combination of the cortical area (forelimb or hindlimb) and the spinal cord level (cervical or lumber), however there was no significant difference among them. In some cells biocytin solution was injected through a recording electrode after EPSP was recorded. One third of them were thought to be the spinal motoneurons from their morphology. To our knowledge, this is the frst work to reconstruct the corticospinal projection with functioning synapses in the coculture preparation. This would be a useful in vitro system for studying normal development and pa! hophysiology of the system.