Until recently, investigations of the neurobiological substrates of simple forms of learning and memory in the marine snail Aplysia have focused mostly on plastic changes that occur within the presynaptic sensory neurons. Here, I summarize the results of recent studies that indicate that exclusively presynaptic processes cannot account for simple forms of learning in Aplysia. In particular, I present evidence that postsynaptic mechanisms play a far more important role in nonassociative learning in Aplysia than has been appreciated before now. Moreover, I describe recent data that suggests the intriguing hypothesis that the persistent, learning-induced changes in Aplysia sensory neurons might depend critically on postsynaptic signals for their induction. Finally, I discuss the potential applicability of this hypothesis to learning-related synaptic plasticity in the mammalian brain.

Zebrafish (Danio rerio) hold immense promise for the study of learning and memory. Several attractive properties characterize this vertebrate animal model. For instance, zebrafish possesses a relatively reduced neural circuitry, which facilitates the drawing of causal relationships between changes on the behavioral scale to changes on the cellular-molecular level. They can also be affected by and absorb pharmacological agents in bath, and undergo learning and memory paradigms as early as 5 days post-fertilization (dpf). In particular, following a brief introduction of a noxious stimulus, allyl isothiocyanate (mustard oil, MO), 5-6 dpf zebrafish exhibit short-term sensitization. This memory has been demonstrated behaviorally through increased thigmotaxis and locomotion. However, the neuromodulatory mechanisms behind this non-declarative, non-associative form of memory remains to be elucidated. Exploiting this animal model’s strengths, we placed zebrafish under high-throughput pharmacological dissections with dopamine-receptor antagonists SCH-23390, eticlopride hydrochloride, haloperidol, and L-745,870 trihydrochloride in order to determine the role of dopamine and delineate receptor subtype-specific influences. These blockades have not only confirmed the role of dopamine, but also have demonstrated a differential involvement of the receptor subtypes within the two behaviors. Nonetheless, further investigations are required in order to determine the influence of other neuromodulators in the establishment of MO-induced short-term sensitization.

Protein synthesis has been shown to be crucial in learning and memory. Particularly, the activity of protein kinase C (PKC) is necessary for long-term memory. Previous studies have shown that serotonin pathway had a profound effect on PKC, and it facilitates long-term potentiation. To study the fundamental molecular processes involved, Aplysia was used to evaluate the changes in RNA expression levels. Ganglia-treated samples as well as behaviorally trained samples were used to analyze any differences after training and the use of drugs, chelerythrine, a selective PKC inhibitor, and Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor. Application of these drugs may reveal different regulatory mechanisms that govern transcription level changes, ultimately leading to protein synthesis. Although the results did not show statistical significance, the pattern of slightly increased RNA level of PKC and calpain after serotonin or behavior training show there is a possible correlation between long-term memory and nuclear regulation of protein synthesis. In addition, because TSA was able to reverse the effects of chelerythrine, there is an indication that an epigenetic regulation may be involved in memory. However, molecular analysis demonstrated inconclusive data, suggesting further examinations of the epigenetic changes exhibiting a brief cellular response.

Zebrafish (Danio rerio) holds a promise as a model vertebrate system for neurobiological investigations of learning and memory. Here, we sought to demonstrate two forms of nonassociative learning: first, short-term sensitization of tail movements and second, long-term sensitization of thigmotaxis, induced by application of allyl isothiocyanate, a chemical irritant, in both restrained and free-swimming larval zebrafish. We found that allyl isothiocyanate increased both tail movements and tail movements’ duration for several minutes after the chemical irritant was removed from the bath. Moreover, pharmacological manipulations were observed to alter the behavior. Experimental manipulations included exposure to methiothepin, an antagonist of serotonin (5-HT) receptors. Methiothepin application was observed to block MO-induced sensitization of thigmotaxis in larval zebrafish. Our results demonstrate that a noxious stimulus, allyl isothiocyanate, can reliably induced tail movements and thigmotaxis sensitization in both restrained and free-swimming larval zebrafish, and thus facilitate future investigations of in vivo neural circuits.

The larval form of the zebrafish (Danio rerio) holds significant promise as a model vertebrate system for understanding the neural mechanisms of behavior. This promise arises from two particularly attractive properties of zebrafish larva: their translucence, which permits robust optogenetic manipulations, and their possession of a relatively simple neural circuitry, which facilitates cellular analysis of behavior. In particular, past studies successfully demonstrated behavioral changes in fish as young as five days post fertilization (DPF) via transient receptor potential A1 (TRPA1) ion channel activation mediated by the chemical irritant allyl isothiocyanate (mustard oil - MO). In the present study, we found that 10 �M total bath concentration of MO significantly increased the duration of tail activity responses in 5 and 6 DPF, agarose-restrained larval zebrafish compared to control treated animals. Further, we recapitulated this sensitizing effect in agarose-restrained older fish, 12 DPF, and to our knowledge, uniquely, in fish as young as 3 DPF. Together, these results indicate the immense promise that zebrafish hold as a neurobiologically tractable animal model for the study of learning and memory, especially non-associative memory.

Chronic pain and anxiety disorders are known to be intimately connected in humans. Occasionally, the sensation of pain and the associated emotional affects like anxiety linger past the end of the triggering stimulus or even in the absence of a trigger. Central sensitization has been posited as a simple form of memory that may underlie the persistence of idiopathic disorders like fibromyalgia. In the present study, we examined the effect of a known chemical irritant, allyl isothiocyanate (mustard oil), and electric shock on freely swimming zebrafish larvae (5 and 12 DPF). We found that mustard oil (MO, concentration = 10�M) and electric shock induced thigmotaxis, an anxiety-related behavior. Future work should determine the specific changes induced by MO or shock in the behavioral circuits that mediate swimming in the zebrafish. The results of this future work may contribute to our understanding of the neural basis of pain and anxiety disorders.

Sudden cardiac death is one of the major leading causes of death in the United States, affecting about 300,000 people annually on average. Cardiac arrhythmias and ventricular fibrillation can be triggered, at the cellular level, by the presence of aberrations of the cardiac action potential (AP) known as early afterdepolarizations (EADs). EADs are single or multiple voltage oscillations largely induced by the reactivation of L-type Ca2+ currents (ICa,L) during phase 2 and phase 3 of a cardiac AP. Our recent studies using dynamic clamp techniques have suggested that EADs and their arrhythmogenic consequences can be potently suppressed by subtle reduction the ICa,L current non-inactivating (pedestal) component and/or minimal changes (3-5 mV) in the voltage dependence of activation. Exploiting the modulatory effects of L-type Ca2+ channel (LTCC) auxiliary β2 subunits on the non-inactivating component of ICa,L, we sought to investigate the effects of knocking down Cavβ2 subunit expression levels in rabbit ventricular myocytes in the presence of an oxidative stress known to trigger EADs (H2O2). We hypothesized that reducing the expression level of endogenous Cavβ2 decreases the probability of EAD occurrence in cardiomyocytes exposed to H2O2. Using an adenoviral infection to deliver a short hairpin RNA (shRNA) specific for targeting Cavβ2 that inhibits its gene expression by binding to its mRNA transcripts, our results showed that myocytes expressing less Cavβ2 mRNA exhibited no EADs; whereas, the control myocytes infected with GFP alone as a control group were more susceptible to EAD occurrence in 0.6 mM H2O2. These results suggest that Cavβ2 could be a potential target for gene therapy and could give insights to other therapeutic strategies that could possibly be implemented.