Physical and comparative mapping of distal mouse chromosome 16. 5 p5.

Distal mouse Chromosome 16 (Chr. 16) includes a region of conserved linkage with human Chromosome 21 (Chr. 21). Mouse models of Down syndrome based on trisomy of distal Chr. 16 have several phenotypes similar to those seen in human patients and have proven useful for correlating dosage imbalance of specific genes with specific developmental anomalies. The degree to which such findings can be related to Down syndrome depends on how well the conserved synteny is maintained. Twenty-four genes have been mapped in both species and there are no discordancies, but the region could carry hundreds of genes. Comparative sequence represents the ultimate comparative map and will aid in identification of genes and their regulatory sequences. A physical map of the distal 4.5 Mb of Chr. 16 has been assembled as an essential step toward a map of sequence-ready templates. The map consists of 51 YACs and 15 BACs and includes 18 transcripts, 9 of which are mapped for the first time in mouse, and 3 of which are, for the first time, described in either species. YAC fragmentation was used to precisely localize the 49 markers on the map. Comparison of this physical map with that of the corresponding region on Chr. 21 shows conservation not only of gene order but of size in the 3 Mb from Cbr1 to Ets2; distal to Ets2, the human map is expanded.

Assessment of a mutation in the H5 domain of Girk2 as a candidate for the weaver mutation.

A mutation in the GIRK2 inwardly rectifying K+ channel was mapped recently to the region of mouse chromosome 16 containing the wv gene and shown to occur in mutant but not in wild-type mice. We demonstrate tight linkage of the Girk2 mutation to the wv phenotype and refine the localization of the weaver (wv) gene on recombinational and physical maps. This linkage between Girk2 and wv has existed since at least 1988 in descendants of the original mutation maintained in C57BL/6 animals. Girk2 is shown to be transcribed in brain before the first recognized manifestation of the wv phenotype and in cultures of granule cells (GCs) isolated from cerebellum at postnatal day 8. Wild-type GCs grown in this culture system display an important developmental property--the ability to extend neurites. However, no inwardly rectifying K+ current is detected in GCs cultured from either wv/wv or +/+ cerebellum under a variety of conditions that activate related channels in other tissues. This suggests that if the Girk2 mutation is responsible for the wv phenotype, it does not act by altering these electrical properties of developing GCs.

High-resolution mapping of D16led-1, Gart, Gas-4, Cbr, Pcp-4, and Erg on distal mouse chromosome 16.

More than 500 backcross progeny from four intersubspecific backcrosses were typed for six markers on distal mouse chromosome 16. Five of these represented genes that mapped within the Sod-1 to Ets-2 interval, which was shown previously to contain the weaver (wv) gene. The map order, including previously mapped reference markers, is (cen)-D16H21S16-D16Led-1-App-Sod-1-Gart-Gas-4-Cbr++ +-wv-Pcp-4-Erg-Ets-2. This gene order recapitulates the order of the genes on human chromosome 21 where known. Two of these markers further define the region containing the weaver gene to a 3.9-cM segment between Cbr and Pcp-4. In addition, Pcp-4 was localized to human chromosome 21 by the presence of a human-specific restriction fragment in WAV-17, a mouse-human somatic cell hybrid with human chromosome 21 as the only human contribution.

Effects of unilateral climbing fibre deafferentation on cytochrome oxidase activity in the developing rat cerebellum.

In a previous study, we found a relationship between climbing fibre synaptogenesis and cytochrome oxidase activity in Purkinje cells during normal development of the rat cerebellum. To determine whether removal of a major depolarizing afferent would alter the level of cytochrome oxidase activity in a post-synaptic neuron, climbing fibre input to Purkinje cells in the right hemicerebellum was interrupted by unilateral pedunculotomy in postnatal day 1 rat pups. After survival to postnatal day 5 (P5) or postnatal day 10 (P10), the cytochrome oxidase reactivity of mitochondria, packing density of mitochondria and perikaryal area of Purkinje cell somata were quantified at the electron microscopic level and compared with the same parameters in both sham-operated animals and normal controls. We found that the areal and numerical densities of darkly reactive mitochondria were lower in deafferented cells than those in the sham-operated animals. Cells of sham-operated animals, however, had higher densities of darkly reactive mitochondria than those in normal animals of the same age group, indicating that cell shrinkage or retarded growth had an effect on the levels of cytochrome oxidase activity in the operated animals. In addition, both operated groups had higher numerical densities of mitochondria than cells of normal animals, reflecting the decreased cell size of the sham and deafferented groups. From these data, we concluded that neonatal destruction of climbing fibres leads to lower levels of cytochrome oxidase activity in Purkinje cell somata that survived to both P5 and P10. The data from the P5 animals was more striking than that from P10, perhaps reflecting the increased number of synaptic interactions of Purkinje cells at P10. We also concluded that destruction of excitatory input did not lead to changes in the total area or number of mitochondria in a post-synaptic neuron, indicating that there was a conversion from darkly to lightly reactive mitochondria in the partially deafferented neurons; however, this may also reflect the smaller cell size of the deafferented group. Thus, our results further substantiate the close relationship between the levels of cytochrome oxidase activity in Purkinje cell somata and the type of input that they receive or fail to receive.

Relationship between synaptogenesis and cytochrome oxidase activity in Purkinje cells of the developing rat cerebellum.

In the adult CNS, the level of oxidative metabolism, as indicated by cytochrome oxidase cytochemistry, can be correlated with the level of neuronal activity. Specifically, heightened cytochrome oxidase activity in post-synaptic neurons can often be correlated with a greater proportion of excitatory inputs, whereas inhibitory inputs often result in a low level of cytochrome oxidase activity. This relationship has not been explored in developing neurons. To this end, cytochrome oxidase cytochemistry was used to compare the levels of oxidative metabolism in rat cerebellar Purkinje cells at various stages of their development. The results indicated that the level of cytochrome oxidase activity in Purkinje cell somata and dendrites correlated closely with the type of synaptic input (excitatory or inhibitory) received by the different segments of the cell. When the cell somata received predominantly excitatory input from climbing fibers, their mitochondria were evenly distributed between the three reactive classes: dark, moderate, and lightly reactive for cytochrome oxidase. When the cell somata received predominantly inhibitory input from basket cell terminals, lightly reactive mitochondria were the prevailing type. Further support for the correlation of excitatory synaptic input with high levels of cytochrome oxidase activity was found in the quantitation of mitochondria within Purkinje cell dendrites. These dendrites received largely excitatory input at all ages and had high levels of cytochrome oxidase activity throughout development and adulthood. There was also a relationship between the level of cytochrome oxidase activity and mitochondrial size within Purkinje cell somata and dendrites from birth to adult. Darkly reactive mitochondria had a greater mean area than moderately reactive mitochondria which, in turn, had a greater mean area than lightly reactive mitochondria. In addition, the packing density of mitochondria within the cytoplasm varied with age in both somata and dendrites. In the somata, the packing density peaked at postnatal day 7, and in dendrites, the peak occurred at postnatal day 10. These data indicate that in a developing system, postsynaptic neurons respond to sequential excitatory and inhibitory inputs by sequential heightening and lowering of their energy metabolism. Thus, cytochrome oxidase activity in a postsynaptic neuron can be correlated with the predominant type of synaptic input that it receives.

Double-labeling of rat alpha-motoneurons for cytochrome oxidase and retrogradely transported [3H]WGA.

In this study, we have demonstrated the co-localization of a retrograde tracer and the reaction product of an oxidative enzyme within the same neurons in the same spinal cord section, using [3H] wheat germ agglutinin and cytochrome oxidase histochemistry. This approach allows unequivocal identification of the alpha-motoneurons innervating specific muscles. We have determined that there is a positive correlation between the distribution of cytochrome oxidase reactivity in alpha-motoneurons and the muscles that they innervate. The degree of cytochrome oxidase reactivity within the labeled alpha-motoneurons appears to be independent of spinal cord level and cell size.