Confocal microscopy of a newly identified protein associated with heart development in the Mexican axolotl.

Recessive mutant gene c for "'cardiac nonfunction" in the mexican axolotl, Ambystoma mexicanum, results in a failure of affected embryos to develop contracting hearts. Mutant embryos survive approximately 4 weeks after fertilization, but eventually die from a lack of circulation. Morphological studies show that mutant hearts lack organized sarcomeric myofibrils. This abnormality can be corrected by co-culturing early mutant hearts with normal anterior endoderm/mesoderm tissues, by culturing them in a medium "conditioned" by this normal tissue, or by RNA isolated from normal endoderm/mesoderm. Additionally, RNA isolated from normal anterior endoderm/mesoderm conditioned medium corrects the mutant hearts in a dose-dependent manner. A cDNA library is constructed using this RNA. On the basis of sequence analyses on this cDNA library, it was estimated that 56% of the total RNA present in the conditioned medium is rRNA, while 44% is nonribosomal RNA. One of the nonribosomal RNAs that showed no significant homology with other known sequences in the Genebank was examined further. An RT-PCR analysis showed that this RNA (designated "N1") is expressed in juvenile skeletal muscle, brain, and heart in significant amounts, less in the lung and not at all in the liver tissue. Affinity-purified polyclonal antipeptide antibodies were produced against the most antigenic portion of the polypeptide which was deduced from this RNA. Western blot analyses of adult heart homogenates, using these antibodies, showed a specific doublet staining at 67 kDa and 65 kDa. These doublets were purified and analyzed for their amino acid composition which showed that both bands most likely belong to the same protein. The N1-protein was further investigated to determine its localization in normal isolated hearts at embryonic stages 35, 38, and 41 and on cross-sections through the heart regions of whole normal embryos at stages 16, 33-34, 37-38, and 41-42 using immunohistochemical techniques and confocal microscopy. In addition, mutant embryos at stage 37-38 were studied for the presence and distribution of the N1-protein on cross-sections through their heart regions. The N1-protein staining was significantly reduced in mutant hearts when compared to normal.

Visual learning suppressed by cooling the temporal pole.

Three monkeys were trained to remember colored photographs of objects over delays of 0, 15, 30, and 45 s. Then two pairs of cooling devices were implanted bilaterally over the anterior 9 mm of the temporal lobe. The devices consisted of 3 X 10 mm loops of stainless steel tubing into which cooled methanol could be pumped. One pair (anterior pair) covered the medial part of the temporal tip (area TG), starting at the rhinal sulcus and extending 3 mm laterally. The second pair (posterior pair) was placed 3 mm lateral to the anterior pair, covering the rest of TG and the anterior extreme of the inferotemporal gyri, anterior TE. Cooling either pair of probes produced a deficit at all delays, but the deficit was greater at the longest delays. There was no difference between cooling the anterior pair and cooling the posterior pair except that cooling the anterior pair greatly increased the disruption of recall that is produced by an interfering stimulus. When all four probes were cooled, which suppressed the function of the entire temporal tip, performance dropped to chance at all delays. While under this condition, the animals could not learn new visual discriminations but could perform previously learned visual discriminations. These results are consistent with the suggestion that the temporal pole is the store for the brief anterograde memory that is available to the medial temporal amnesics.