CREB in the pond snail <i>Lymnaea stagnalis</i>: Cloning, gene expression, and function in identifiable neurons of the central nervous system

Self Cite

Environmentally relevant stressors alter the memory-forming process in Lymnaea following operant conditioning of aerial respiration. One such stressor is heat. Previously, we found that following a 1 h heat shock, long-term memory (LTM) formation was enhanced. We also had shown that the heat stressor activates at least two heat shock proteins (HSPs): HSP40 and HSP70. Here, we tested two hypotheses: (1) the production of HSPs is necessary for enhanced LTM formation; and (2) blocking DNA methylation prevents the heat stressor-induced enhancement of LTM formation. We show here that the enhancing effect of the heat stressor on LTM formation occurs even if snails experienced the stressor 3 days previously. We further show that a flavonoid, quercetin, which inhibits HSP activation, blocks the enhancing effect of the heat stressor on LTM formation. Finally, we show that injection of a DNA methylation blocker, 5-AZA, before snails experience the heat stressor prevents enhancement of memory formation.

Section: Discussionmentioning

confidence: 92%

Heat stress enhances LTM formation inLymnaea: role of HSPs and DNA methylation

Sunada

Riaz

Freitas

et al. 2016

Self Cite

“…However, because the use of the typical protein synthesis inhibitors is not feasible for the ethical treatment of humans, researchers examined other possible pharmacological agents. One such agent was the beta-blocker propranolol, as it is probable that the adrenergic system is involved in at least certain aspects of the stressful event and also because propranolol may inhibit noradrenergicstimulated CREB phosphorylation, a necessary pathway involved in memory consolidation (Thonberg et al, 2002;Sadamoto et al, 2003;Azami et al, 2006). Systemic administration of propranolol in conjunction with memory reactivation disrupts reconsolidation of LTM in auditory and contextual fear and avoidance in rodents (Abrari et al, 2008;Debiec and Ledoux, 2004;Debiec et al, 2011;Przybyslawski et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

Sequential exposure to a combination of stressors blocks memory reconsolidation in Lymnaea

Dodd

Lukowiak

2015

Self Cite

Stress alters the formation of long-term memory (LTM) in Lymnaea. When snails are exposed to more than one stressor, however, how the memory is altered becomes complicated. Here, we investigated how multiple stressors applied in a specific pattern affect an aspect of memory not often studied in regards to stress -reconsolidation. We hypothesized that the application of a sequence of stressors would block the reconsolidation process. Reconsolidation occurs following activation of a previously formed memory. Sequential crowding and handling were used as the stressors to block reconsolidation. When the two stressors were sequentially presented immediately following memory activation, reconsolidation was blocked. However, if the sequential presentation of the stressors was delayed for 1 h after memory activation, reconsolidation was not blocked. That is, LTM was observed. Finally, presentation of either stressor alone did not block reconsolidation. Thus, stressors can block reconsolidation, which may be preferable to pharmacological manipulations.

“…Lymnaea CREB1 and CREB2 mRNAs and salmon GTH α2 RNA The nucleotide sequences of Lymnaea CREB1 and CREB2 mRNAs (accession numbers: AB041522 and AB083656, respectively; for details, see Sadamoto et al, 2004b) were used to design the primers and probes for qRT-PCR. We used the Primer Express software (version 1.0; PE Applied Biosystems) to design appropriate primers and fluorogenic probes for qRT-PCR assays.…”

Section: Preparation Of Primers Probes and Standard Curves Formentioning

confidence: 99%

Determination of the exact copy numbers of particular mRNAs in a single cell by quantitative real-time RT-PCR

Wagatsuma

Sadamoto

Kitahashi

et al. 2005

Self Cite

SUMMARY Gene expression is differently regulated in every cell even though the cells are included in the same tissue. For this reason, we need to measure the amount of mRNAs in a single cell to understand transcription mechanism better. However, there are no accurate, rapid and appropriate methods to determine the exact copy numbers of particular mRNAs in a single cell. We therefore developed a procedure for isolating a single, identifiable cell and determining the exact copy numbers of mRNAs within it. We first isolated the cerebral giant cell of the pond snail Lymnaea stagnalis as this neuron plays a key role in the process of memory consolidation of a learned behavior brought about by associative learning of feeding behavior. We then determined the copy numbers of mRNAs for the cyclic AMP-responsive element binding proteins (CREBs). These transcription factors play an important role in memory formation across animal species. The protocol uses two techniques in concert with each other: a technique for isolating a single neuron with newly developed micromanipulators coupled to an assay of mRNAs by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The molecular assay determined the mRNA copy numbers, each of which was compared with a standard curve prepared from cDNA solutions corresponding to the serially diluted solutions of Lymnaea CREB mRNA. The standard curves were linear within a range of 10 to 105 copies, and the intra-assay variation was within 15%. Each neuron removed from the ganglia was punctured to extract the total RNA directly and was used for the assay without further purification. Using this two-step procedure, we found that the mRNA copy number of CREB repressor (CREB2) was 30–240 in a single cerebral giant cell, whereas that of CREB activator (CREB1) was below the detection limits of the assay (<25). These results suggest that the CREB cascade is regulated by an excess amount of CREB2 in the cerebral giant cells. Our procedure is the only quantitative analysis for elucidation of the dynamics of gene transcription in a single cell.