Training-dependent associative learning induced neocortical structural plasticity: a trace eyeblink conditioning analysis.

Studies utilizing general learning and memory tasks have suggested the importance of neocortical structural plasticity for memory consolidation. However, these learning tasks typically result in learning of multiple different tasks over several days of training, making it difficult to determine the synaptic time course mediating each learning event. The current study used trace-eyeblink conditioning to determine the time course for neocortical spine modification during learning. With eyeblink conditioning, subjects are presented with a neutral, conditioned stimulus (CS) paired with a salient, unconditioned stimulus (US) to elicit an unconditioned response (UR). With multiple CS-US pairings, subjects learn to associate the CS with the US and exhibit a conditioned response (CR) when presented with the CS. Trace conditioning is when there is a stimulus free interval between the CS and the US. Utilizing trace-eyeblink conditioning with whisker stimulation as the CS (whisker-trace-eyeblink: WTEB), previous findings have shown that primary somatosensory (barrel) cortex is required for both acquisition and retention of the trace-association. Additionally, prior findings demonstrated that WTEB acquisition results in an expansion of the cytochrome oxidase whisker representation and synaptic modification in layer IV of barrel cortex. To further explore these findings and determine the time course for neocortical learning-induced spine modification, the present study utilized WTEB conditioning to examine Golgi-Cox stained neurons in layer IV of barrel cortex. Findings from this study demonstrated a training-dependent spine proliferation in layer IV of barrel cortex during trace associative learning. Furthermore, findings from this study showing that filopodia-like spines exhibited a similar pattern to the overall spine density further suggests that reorganization of synaptic contacts set the foundation for learning-induced neocortical modifications through the different neocortical layers.

Elevated Arc/Arg 3.1 protein expression in the basolateral amygdala following auditory trace-cued fear conditioning.

The underlying neuronal mechanisms of learning and memory have been heavily explored using associative learning paradigms. Two of the more commonly employed learning paradigms have been contextual and delay fear conditioning. In fear conditioning, a subject learns to associate a neutral stimulus (conditioned stimulus; CS), such as a tone or the context of the room, with a fear provoking stimulus (unconditioned stimulus; US), such as a mild footshock. Utilizing these two paradigms, various analyses have elegantly demonstrated that the amygdala plays a role in both fear-related associative learning paradigms. However, the amygdala's involvement in trace fear conditioning, a forebrain-dependent fear associative learning paradigm that has been suggested to tap into higher cognitive processes, has not been closely investigated. Furthermore, to our knowledge, the specific amygdala nuclei involved with trace fear conditioning has not been examined. The present study used Arc expression as an activity marker to determine the amygdala's involvement in trace fear associative learning and to further explore involvement of specific amygdalar nuclei. Arc is an immediate early gene that has been shown to be associated with neuronal activation and is believed to be necessary for neuronal plasticity. Findings from the present study demonstrated that trace-conditioned mice, compared to backward-conditioned (stimulation-control), delay-conditioned and naïve mice, exhibited elevated amygdalar Arc expression in the basolateral (BLA) but not the central (CeA) or the lateral amygdala (LA). These findings are consistent with previous reports demonstrating that the amygdala plays a critical role in trace conditioning. Furthermore, these findings parallel studies demonstrating hippocampal-BLA activation following contextual fear conditioning, suggesting that trace fear conditioning and contextual fear conditioning may involve similar amygdala nuclei. Together, findings from this study demonstrate similarities in the pathway for trace and contextual fear conditioning, and further suggest possible underlying mechanisms for acquisition and consolidation of these two types of fear-related learning.

Neocortical synaptic proliferation following forebrain-dependent trace associative learning.

Many behavioral studies have suggested that learning induces neocortical synaptic modifications. However, neocortical synaptic modifications following forebrain-dependent trace associative learning has not been closely examined. Acquisition of whisker-trace-eyeblink (WTEB) conditioning, a forebrain-dependent trace associative task, has been reported to modulate the expression of cytochrome oxidase, a marker for metabolic activity, in the conditioned barrels, suggesting that trace associative conditioning induces neocortical synaptic plasticity. However, neocortical synaptic plasticity has never been directly examined following this trace associative task. To assess neocortical synaptic modifications, the present study examined synapsin I expression following WTEB conditioning. Synapsin I is part of a phosphoprotein family involved in neuronal regulation of neurotransmitter release that also exhibits an expression pattern closely correlating to synapse number. Findings from this study demonstrated that synapsin I expression is elevated in primary somatosensory neocortex in trace-paired-conditioned mice compared with unpaired-conditioned (stimulation-control) mice and naïve mice, suggesting that WTEB conditioning induces synaptic proliferation. Additional findings from the present study examining cytochrome oxidase expression replicated previous findings demonstrating that WTEB conditioning induces a learning-specific expansion of the cytochrome oxidase staining expression for conditioned barrels. Together, these results suggest that synaptic proliferation is contributing to the learning-induced metabolic augmentation previously observed in conditioned barrels following WTEB conditioning. Furthermore, these results suggest that trace associative learning facilitates neocortical synaptic modification.

Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulators.

Kinesins are a group of related molecular motor proteins that have great potential as targets for antimitotic drug development. We have developed two novel assays, one end-point and one kinetic, that are useful for the discovery and optimization of kinesin modulators. Both assays measure inorganic phosphate (Pi) generated by microtubule-activated kinesin adenosine triphosphatase activity. The assays were validated using the mitotic Eg5 kinesin-specific inhibitor, monastrol. A panel of nine kinesin motor domain proteins, representing 8 of the 14 classes of kinesins, was screened. The coefficient of variation for both assays was determined to be 4-14% depending on the panel member. Using the Eg5 kinetic assay with monastrol the IC50 value was 12 microM, which agrees well with previously published results. Two other closely related mitotic kinesins (AnBimC and MKLP1) were found to have IC50 values in the millimolar range. The other panel members (kinesin heavy chain, chromokinesin KIF4A, KIF3C, CENP-E, MCAK, and KIFC3) were not significantly inhibited by millimolar levels of monastrol. It is anticipated that screening of the nine-member panel of kinesins in these assays will serve as a platform for the discovery and development of specific kinesin modulators.