In vitro formation of corticospinal synapses in an organotypic slice co-culture.

In order to study biological properties of the corticospinal tract, we have reconstructed this system in an in vitro slice culture preparation. Motor cortex and spinal cord slices, prepared from newborn rats, were co-cultured on pored membranes for 16-24 days. Anterograde labeling with biocytin showed that substantial neural connections had formed between the cortex and spinal cord slices. Retrograde labeling with horseradish peroxidase or 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate demonstrated that the parent cells were located primarily in the deeper layer of the cortex, as is found in vivo. Stimulation of the deep layer of the cortex elicited extracellular postsynaptic responses and intracellular excitatory postsynaptic potentials (EPSPs) in the co-cultured spinal cord that were mediated by the 1-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/ kainate-type glutamate receptor. The intracellular injection of biocytin after EPSPs were recorded showed that one-third of these cells were large stellate cells, which are thought to be motoneurons, while a large portion of the remaining labeled cells were bipolar cells of smaller sizes. Using this reconstructed in vitro preparation, we recorded field EPSPs (fEPSPs) along a 100-microm-interval lattice in the spinal gray matter, which allowed the quantitative evaluation of synapse formation. The fEPSP amplitudes were more than two-fold larger when the forelimb cortex was co-cultured with cervical cord rather than lumbar cord. However, hindlimb cortex did not show this preference. The fEPSP amplitudes were more than twice as large when the dorsal side of the spinal cord was adjacent to the cortex than the ventral side. In summary, we have reconstructed the corticospinal projection and synapses in vitro using cortical and spinal explants. This system allows for an efficient quantitative evaluation of synapse formation and for studies of postsynaptic cells. Our results suggest that synapse formation shows preferences along and perpendicular to the neuraxis of the spinal cord.

Multiplication of attenuated and virulent porcine parvoviruses in colostrum-deprived, neonatal pigs.

Colostrum-deprived, neonatal, 2 days old pigs were inoculated with the attenuated HT-/SK or the virulent 90HS strain of porcine parvovirus (PPV) by the oral or subcutaneous route and sacrificed 2, 4 or 6 days after inoculation. Then, comparison was made on viral multiplication in pigs between the two strains. Pigs inoculated with the HT-/SK strain showed no detectable viremia or HI antibody responses against PPV within 6 days after inoculation. Only in pigs inoculated by the subcutaneous route, a small amount of virus was recovered from the spleen, liver, or mesenteric lymph nodes. These viruses were distinguished from the parental virulent 90HS strain, as examined for rct maker in vitro. When pigs were inoculated with the virulent 90HS strain, viremia appeared in all of them 1 day after inoculation and continued for up to the sacrificed day. Moreover, a considerable amount of virus was also detected from all tissues, including brain, lung, liver, spleen, pancreas, small intestine, and lymph node tissues, in all pigs tested. HI antibodies were first detected 6 days after inoculation.