Cellular and molecular analyses of associative learning and memory are the research interests of this laboratory. The experimental preparation we use to study mechanisms of Pavlovian conditioning is the Pacific nudibranch Hermissenda crassicornis. Cellular neurophysiological, biophysical and biochemical studies of the isolated central nervous system of conditioned Hermissenda have identified long-term intrinsic modifications of identified sensory neurons and interneurons in the neural circuit supporting the generation of the conditioned response (CR). We have found that conditioning produces an enhancement of the excitability of identified sensory neurons and interneurons elicited by the conditioned stimulus. In addition, conditioning produces a modification of synaptic connections between identified sensory neurons and interneurons in the neural circuit producing the CR.
Hermissenda is an attractive preparation for studies of molecular mechanisms underlying memory consolidation since behavior can be modified by one conditioning trial and intrinsic cellular/molecular changes can be studied in identified neurons that previously have been shown to contribute to long-term memory produced by Pavlovian conditioning. Molecular mechanisms underlying the formation of memory over-time for this example of associative learning are currently under investigation using a proteomic analysis in conjunction with mass spectrometry (MS). Recent MS studies have identified proteins that express changes in abundance and post-translational modifications regulated by conditioning. As an example, one-trial in vitro conditioning results in the development of an intermediate phase of memory that is characterized by enhanced cellular excitability and the phosphorylation of CSP24, a member of the family of β-thymosin-like actin-binding proteins. Inhibition of CSP24 protein expression blocks the development of intermediate-term memory in Hermissenda. The phosphorylation and abundance of CSP24 are also regulated by multi-trial Pavlovian conditioning. The mechanisms underlying reorganization of the cytoskeleton supporting the formation of memory associated with Pavlovian conditioning of Hermissenda are currently under investigation.
Proteomics is the systematic study of the expression, structure, and function of proteins within cells. The application of proteomic approaches to the study of memory formation holds promise in identifying specific proteins that exhibit altered expression and post-translational modifications with learning. An important feature of proteomics is the separation of complex mixtures of proteins in samples into individual components, typically with 2Dgel electrophoresis (2DE), for subsequent identification with mass spectrometry. Difference gel electrophoresis (DIGE) was developed to overcome the problem of reproducibility between gels inherent with traditional 2DE approaches. DIGE provides for multiplexing, where two samples are labeled with a different fluorescent dye prior to running the two samples on the same gel. The fluorescent dyes used in DIGE, Cy3 and Cy5, are cyanine based, molecular-weight matched, amine reactive and positively charged. These characteristics result in no electrophoretic mobility shifts when the samples are co-electrophoresed resulting in every identical protein in one sample superimposing with its differentially labeled counterpart in the other sample. 2D-DIGE gels labeled with Cy3 from a control group (A), Cy5 from an experimental group (B), and the overlay of the two images (C). Protein spots in regions of equal abundance appear yellow.
Crow, T., Redell, J., Xue-Bian, J.J., Tian, L.-M. and Dash, P. (2003) Inhibition of CSP24 protein expression blocks the development of intermediate-term memory in Hermissenda. Journal of Neuroscience, 23: 3415-3422.
Yamoah, EN, Levic, S, Redell, J, Crow, T. (2005) Inhibition of conditioned stimulus pathway phosphoprotein 24 expression blocks the reduction in A-type transient K+ current produced by one-trial in vitro conditioning of Hermissenda. Journal of Neuroscience, 25: 4793-4800.
Crow, T. and Xue-Bian, J.-J. (2007) One-trial in vitro conditioning of Hermissenda regulates phosphorylation of Ser-122 of Csp24. Ann NY Acad Sci , 1112: 189-200.
Redell, J.B., Xue-Bian, J.J., Bubb, M.R. and Crow, T. (2007) One-trial in vitro conditioning regulates an association between the β-thymosin repeat protein Csp24 and actin. Neuroscience, 148: 413-420.
Crow, T., Xue-Bian, J.-J., and Tian, L.-M. (2009) Learning and memory in invertebrates: Hermissenda. In, New Encyclopedia of Neuroscience, L.R. Squire, ed., Elsevier, Oxford, vol. 5, pp. 427-433.
Jin, N., Tian, L.-M. and Crow, T. (2009) 5-HT and GABA modulate intrinsic excitability of type I interneurons in Hermissenda. Journal of Neurophysiology, 102:2825-2833.
Crow, T., Tian, L.-M. and Xue-Bian, J.J. (2010) Mechanisms of memory formation in Hermissenda. In, Encyclopedia of Behavioral Neuroscience, volume 2 pp. 192-199 G.F. Koob, M. Le Moal, and R.F. Thompson, eds., Elsevier, Oxford.
Crow, T. and Xue-Bian, J.-J. (2010) Proteomic analysis of post-translational modifications in conditioned Hermissenda. Neuroscience, 165:1182-1190.
Crow, T. and Xue-Bian, J.J. (2011) Proteomic analysis of short-and intermediate-term memory in Hermissenda. Neuroscience, 192: 102-111.
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