C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5

Genome-wide Functional Analysis of CREB/Long-Term Memory-Dependent Transcription Reveals Distinct Basal and Memory Gene Expression Programs

SummaryInduced CREB activity is a hallmark of long-term memory, but the full repertoire of CREB transcriptional targets required specifically for memory is not known in any system. To obtain a more complete picture of the mechanisms involved in memory, we combined memory training with genome-wide transcriptional analysis of C. elegans CREB mutants. This approach identified 757 significant CREB/memory-induced targets and confirmed the involvement of known memory genes from other organisms, but also suggested new mechanisms and novel components that may be conserved through mammals. CREB mediates distinct basal and memory transcriptional programs at least partially through spatial restriction of CREB activity: basal targets are regulated primarily in nonneuronal tissues, while memory targets are enriched for neuronal expression, emanating from CREB activity in AIM neurons. This suite of novel memory-associated genes will provide a platform for the discovery of orthologous mammalian long-term memory components

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxyfluorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis

Should patients with abnormal liver function tests in primary care be tested for chronic viral hepatitis: cost minimisation analysis based on a comprehensively tested cohort

Background Liver function tests (LFTs) are ordered in large numbers in primary care, and the Birmingham and Lambeth Liver Evaluation Testing Strategies (BALLETS) study was set up to assess their usefulness in patients with no pre-existing or self-evident liver disease. All patients were tested for chronic viral hepatitis thereby providing an opportunity to compare various strategies for detection of this serious treatable disease. Methods This study uses data from the BALLETS cohort to compare various testing strategies for viral hepatitis in patients who had received an abnormal LFT result. The aim was to inform a strategy for identification of patients with chronic viral hepatitis. We used a cost-minimisation analysis to define a base case and then calculated the incremental cost per case detected to inform a strategy that could guide testing for chronic viral hepatitis. Results Of the 1,236 study patients with an abnormal LFT, 13 had chronic viral hepatitis (nine hepatitis B and four hepatitis C). The strategy advocated by the current guidelines (repeating the LFT with a view to testing for specific disease if it remained abnormal) was less efficient (more expensive per case detected) than a simple policy of testing all patients for viral hepatitis without repeating LFTs. A more selective strategy of viral testing all patients for viral hepatitis if they were born in countries where viral hepatitis was prevalent provided high efficiency with little loss of sensitivity. A notably high alanine aminotransferase (ALT) level (greater than twice the upper limit of normal) on the initial ALT test had high predictive value, but was insensitive, missing half the cases of viral infection. Conclusions Based on this analysis and on widely accepted clinical principles, a "fast and frugal" heuristic was produced to guide general practitioners with respect to diagnosing cases of viral hepatitis in asymptomatic patients with abnormal LFTs. It recommends testing all patients where a clear clinical indication of infection is present (e.g. evidence of intravenous drug use), followed by testing all patients who originated from countries where viral hepatitis is prevalent, and finally testing those who have a notably raised ALT level (more than twice the upper limit of normal). Patients not picked up by this efficient algorithm had a risk of chronic viral hepatitis that is lower than the general population

Cellular and genetic regulation of short and intermediate-term memory and the Gq alpha regulated transition to long-term memory in C. elegans

Current evidence suggests that the molecular basis of short-term associative memory (STAM) arises from changes in protein dynamics that increase the strength of synaptic signaling; however, many of the fundamental molecular mechanisms underlying STAM, particularly in C. elegans, remain unknown. Our lab developed a short-term associative memory (STAM) assay in which worms learn to associate food with an odor and remember this association for over one hour. I have found that a single massed training produces short-term memory that is genetically consistent with short-term memory in other organisms. For example, C. elegans STAM requires cAMP signaling and does not depend on CREB, gene transcription, or translation for 30-minute memory. One-hour memory is translation-dependent indicating that this time-point may be a form of intermediate-term associative memory (ITAM). Interestingly, I also find that translation is required for forgetting after massed training. Additionally, we find that STAM can be distinguished from olfactory adaptation. Adaptation mutants show variable responses to STAM training, suggesting that they may differentially employ the same molecular machinery. By testing canonical short-term memory regulators, I have established that C. elegans STAM training can now confidently be used to uncover novel and conserved short and intermediate-term memory regulators. A gain-of-function mutation in the C. elegans Gqá homolog egl-30, inhibits adaptation, increases CREB expression after starvation, and enhances excitatory neurotransmitter release causing among others a hyperactive, loopy phenotype. egl-30 gain-of-function mutants have strikingly extended STAM with memory after STAM training lasting longer than 24 hours vs. 1 hour for wild-type worms. This 24 hr. memory is dependent on CREB, suggesting that the memory has become long-term. We are currently identifying the neurons and downstream genetic regulators involved egl-30 overexpression memory. To our knowledge, this is the first indication that Gqá is directly involved in the consolidation of and transition between short and long-term memory. These findings indicate that C. elegans STAM is a unique learning and memory paradigm that we can use to elucidate conserved mechanisms of associative memory including molecular determinants and cellular dynamics. Indeed I have identified several novel regulators of memory and forgetting, including Gqá, that will contribute to broader cross-species memory research

C. elegans positive olfactory associative memory is a molecularly conserved behavioral paradigm

AbstractWhile it is thought that short-term memory arises from changes in protein dynamics that increase the strength of synaptic signaling, many of the underlying fundamental molecular mechanisms remain unknown.Our lab developed a Caenorhabditis elegans assay of positive olfactory short-term associative memory (STAM), in which worms learn to associate food with an odor and can remember this association for over 1h. Here we use this massed olfactory associative assay to identify regulators of C. elegans short-term and intermediate-term associative memory (ITAM) processes. We show that there are unique molecular characteristics for different temporal phases of STAM, which include: learning, which is tested immediately after training, short-term memory, tested 30min after training, intermediate-term memory, tested 1h after training, and forgetting, tested 2h after training. We find that, as in higher organisms, C. elegans STAM requires calcium and cAMP signaling, and ITAM requires protein translation. Additionally, we found that STAM and ITAM are distinct from olfactory adaptation, an associative paradigm in which worms learn to disregard an inherently attractive odor after starvation in the presence of that odor. Adaptation mutants show variable responses to short-term associative memory training. Our data distinguish between shorter forms of a positive associative memory in C. elegans that require canonical memory pathways. Study of STAM and ITAM in C. elegans could lead to a more general understanding of the distinctions between these important processes and also to the discovery of novel conserved memory regulators

Activation of Gαq Signaling Enhances Memory Consolidation and Slows Cognitive Decline

Perhaps the most devastating decline with age is the loss of memory. Therefore, identifying mechanisms to restore memory function with age is critical. Using C. elegans associative learning and memory assays, we identified a gain-of-function Gαq signaling pathway mutant that forms a long-term (cAMP response element binding protein [CREB]-dependent) memory following one conditioned stimulus-unconditioned stimulus (CS-US) pairing, which usually requires seven CS-US pairings. Increased CREB activity in AIM interneurons reduces the threshold for memory consolidation through transcription of a set of previously identified "long-term memory" genes. Enhanced Gαq signaling in the AWC sensory neuron is both necessary and sufficient for improved memory and increased AIM CREB activity, and activation of Gαq specifically in aged animals rescues the ability to form memory. Activation of Gαq in AWC sensory neurons non-cell autonomously induces consolidation after one CS-US pairing, enabling both cognitive function maintenance with age and restoration of memory function in animals with impaired memory performance without decreased longevity