Cold and Lithium Delay Forgetting of Olfactory Memories in C. elegans

The poet W.B Yeats wrote that “All that is personal soon rots, it must be packed in ice or salt”. Here we show that in Caenorhabditis elegans nematodes, memories are preserved on ice and in lithium salt. We discovered that cold delays forgetting of specific olfactory memories by >8-fold. Adaptation to low temperatures cancels delayed forgetting. To study the underlying mechanism we performed RNA-seq, mutant analyses, and pharmacological assays. We found that regulation of membrane properties switches cold-induced delayed forgetting ON and OFF, and, remarkably, that lithium delays forgetting only in cold-sensitive but not cold-tolerant worms. We found that downregulation of the diacylglycerol pathway in AWC sensory neurons is essential for lithium-mediated delayed forgetting, and that long-term suppression occurs in the downstream AIY interneurons, as shown by neuronal activity recordings. We suggest that the worms' genetic tractability might be harnessed to study how lithium and cold affect the brain, and even more fundamentally, how memory is stored and lost. Overall design: To investigate the genetic basis of delayed forgetting on ice, C. elegans were trained with the odorant butanone and subsequently placed on ice for 2 hours prior to RNA extraction using Trizol. Gene expression profiles were analyzed through mRNA sequencing under six distinct experimental conditions, with three biological replicates per condition. The experimental groups were defined based on cultivation temperature and training regime: (1) 20°C Cultivation group – Worms cultivated at 20°C, representing cold-sensitive individuals that exhibit delayed forgetting when exposed to ice. (2) 15°C Cultivation group – Worms cultivated overnight at 15°C, representing cold-tolerant individuals that do not exhibit delayed forgetting on ice. (3) 15°C to 20°C Temperature-shift group– Worms cultivated overnight at 15°C, then transferred to 20°C for 2 hours, to reverse the cold tolerance acquired from overnight exposure. cDNA was prepared using either the SMART-Seq HT Kit or SMART-Seq v4 Ultra Kit. For all samples, 10 ng of RNA was used as input and processed according to the manufacturer's protocol. Double-stranded cDNA was amplified by 10 PCR cycles for SMART-Seq HT samples and 12 cycles for SMART-Seq v4 Ultra samples. cDNA quantity and quality were verified using the TapeStation 2200 (Agilent). Sequencing libraries were prepared from 1 ng of cDNA using the Nextera XT DNA Library Preparation Kit (Illumina), following the manufacturer's instructions. Library quality and concentration were confirmed using the TapeStation 2200, and libraries were pooled according to molarity for sequencing on the Illumina NextSeq 500 platform.