Innate and learned odor-guided behaviors utilize distinct molecular signaling pathways in a shared dopaminergic circuit.

Odor-based learning and innate odor-driven behavior have been hypothesized to require separate neuronal circuitry. Contrary to this notion, innate behavior and olfactory learning were recently shown to share circuitry that includes the Drosophila mushroom body (MB). But how a single circuit drives two discrete behaviors remains unknown. Here, we define an MB circuit responsible for both olfactory learning and innate odor avoidance and the distinct dDA1 dopamine receptor-dependent signaling pathways that mediate these behaviors. Associative learning and learning-induced MB plasticity require rutabaga-encoded adenylyl cyclase activity in the MB. In contrast, innate odor preferences driven by naive MB neurotransmission are rutabaga independent, requiring the adenylyl cyclase ACXD. Both learning and innate odor preferences converge on PKA and the downstream MBON-γ2α'1. Importantly, the utilization of this shared circuitry for innate behavior only becomes apparent with hunger, indicating that hardwired innate behavior becomes more flexible during states of stress.

Genetics and Molecular Biology of Memory Suppression.

The brain is designed not only with molecules and cellular processes that help to form memories but also with molecules and cellular processes that suppress the formation and retention of memory. The latter processes are critical for an efficient memory management system, given the vast amount of information that each person experiences in their daily activities and that most of this information becomes irrelevant with time. Thus, efficiency dictates that the brain should have processes for selecting the most critical information for storage and suppressing the irrelevant or forgetting it later should it escape the initial filters. Such memory suppressor molecules and processes are revealed by genetic or pharmacologic insults that lead to enhanced memory expression. We review here the predominant memory suppressor molecules and processes that have recently been discovered. They are diverse, as expected, because the brain is complex and employs many different strategies and mechanisms to form memories. They include the gene-repressive actions of small noncoding RNAs, repressors of protein synthesis, cAMP-mediated gene expression pathways, inter- and intracellular signaling pathways for normal forgetting, and others. A deep understanding of memory suppressor molecules and processes is necessary to fully comprehend how the brain forms, stabilizes, and retrieves memories and to reveal how brain disorders disrupt memory.

Associative learning drives longitudinally graded presynaptic plasticity of neurotransmitter release along axonal compartments.

Anatomical and physiological compartmentalization of neurons is a mechanism to increase the computational capacity of a circuit, and a major question is what role axonal compartmentalization plays. Axonal compartmentalization may enable localized, presynaptic plasticity to alter neuronal output in a flexible, experience-dependent manner. Here, we show that olfactory learning generates compartmentalized, bidirectional plasticity of acetylcholine release that varies across the longitudinal compartments of Drosophila mushroom body (MB) axons. The directionality of the learning-induced plasticity depends on the valence of the learning event (aversive vs. appetitive), varies linearly across proximal to distal compartments following appetitive conditioning, and correlates with learning-induced changes in downstream mushroom body output neurons (MBONs) that modulate behavioral action selection. Potentiation of acetylcholine release was dependent on the CaV2.1 calcium channel subunit cacophony. In addition, contrast between the positive conditioned stimulus and other odors required the inositol triphosphate receptor, which maintained responsivity to odors upon repeated presentations, preventing adaptation. Downstream from the MB, a set of MBONs that receive their input from the γ3 MB compartment were required for normal appetitive learning, suggesting that they represent a key node through which reward learning influences decision-making. These data demonstrate that learning drives valence-correlated, compartmentalized, bidirectional potentiation, and depression of synaptic neurotransmitter release, which rely on distinct mechanisms and are distributed across axonal compartments in a learning circuit.

Memory suppressor genes: Modulating acquisition, consolidation, and forgetting.

The brain has a remarkable but underappreciated capacity to limit memory formation and expression. The term "memory suppressor gene" was coined in 1998 as an attempt to explain emerging reports that some genes appeared to limit memory. At that time, only a handful of memory suppressor genes were known, and they were understood to work by limiting cAMP-dependent consolidation. In the intervening decades, almost 100 memory suppressor genes with diverse functions have been discovered that affect not only consolidation but also acquisition and forgetting. Here we highlight the surprising extent to which biological limits are placed on memory formation through reviewing the literature on memory suppressor genes. In this review, we present memory suppressors within the framework of their actions on different memory operations: acquisition, consolidation, and forgetting. This is followed by a discussion of the reasons why there may be a biological need to limit memory formation.

Ras acts as a molecular switch between two forms of consolidated memory in Drosophila.

Long-lasting, consolidated memories require not only positive biological processes that facilitate long-term memories (LTM) but also the suppression of inhibitory processes that prevent them. The mushroom body neurons (MBn) in Drosophila melanogaster store protein synthesis-dependent LTM (PSD-LTM) as well as protein synthesis-independent, anesthesia-resistant memory (ARM). The formation of ARM inhibits PSD-LTM but the underlying molecular processes that mediate this interaction remain unknown. Here, we demonstrate that the Ras→Raf→rho kinase (ROCK) pathway in MBn suppresses ARM consolidation, allowing the formation of PSD-LTM. Our initial results revealed that the effects of Ras on memory are due to postacquisition processes. Ras knockdown enhanced memory expression but had no effect on acquisition. Additionally, increasing Ras activity optogenetically after, but not before, acquisition impaired memory performance. The elevated memory produced by Ras knockdown is a result of increased ARM. While Ras knockdown enhanced the consolidation of ARM, it eliminated PSD-LTM. We found that these effects are mediated by the downstream kinase Raf. Similar to Ras, knockdown of Raf enhanced ARM consolidation and impaired PSD-LTM. Surprisingly, knockdown of the canonical downstream extracellular signal-regulated kinase did not reproduce the phenotypes observed with Ras and Raf knockdown. Rather, Ras/Raf inhibition of ROCK was found to be responsible for suppressing ARM. Constitutively active ROCK enhanced ARM and impaired PSD-LTM, while decreasing ROCK activity rescued the enhanced ARM produced by Ras knockdown. We conclude that MBn Ras/Raf inhibition of ROCK suppresses the consolidation of ARM, which permits the formation of PSD-LTM.

Aversive Training Induces Both Presynaptic and Postsynaptic Suppression in Drosophila.

The α'ß' subtype of Drosophila mushroom body neurons (MBn) is required for memory acquisition, consolidation and early memory retrieval after aversive olfactory conditioning. However, in vivo functional imaging studies have failed to detect an early forming memory trace in these neurons as reflected by an enhanced G-CaMP signal in response to presentation of the learned odor. Moreover, whether cellular memory traces form early after conditioning in the mushroom body output neurons (MBOn) downstream of the α'ß' MBn remains unknown. Here, we show that aversive olfactory conditioning suppresses the calcium responses to the learned odor in both α'3 and α'2 axon segments of α'ß' MBn and in the dendrites of α'3 MBOn immediately after conditioning using female flies. Notably, the cellular memory traces in both α'3 MBn and α'3 MBOn are short-lived and persist for <30 min. The suppressed response in α'3 MBn is accompanied by a reduction of acetylcholine (ACh) release, suggesting that the memory trace in postsynaptic α'3 MBOn may simply reflect the suppression in presynaptic α'3 MBn. Furthermore, we show that the α'3 MBn memory trace does not occur from the inhibition of GABAergic neurons via GABAA receptor activation. Because activation of the α'3 MBOn drives approach behavior of adult flies, our results demonstrate that aversive conditioning promotes avoidance behavior through suppression of the α'3 MBn-MBOn circuit.SIGNIFICANCE STATEMENTDrosophila learn to avoid an odor if that odor is repeatedly paired with electric shock. Mushroom body neurons (MBns) are known to be major cell types that mediate this form of aversive conditioning. Here we show that aversive conditioning causes a reduced response to the conditioned odor in an axon branch of one subtype of the MBn for no more than 30 min after conditioning, and in the dendrites of postsynaptic, MB output neurons (MBOns). Because experimenter-induced activation of the MBOn induces approach behavior by the fly, our data support a model that aversive learning promotes avoidance by suppressing the MBn-MBOn synapses that normally promote attraction.

Regulation of Itch and Nedd4 E3 Ligase Activity and Degradation by LRAD3.

Itch and Nedd4 are members of the Nedd4 family of E3 ubiquitin ligases that are important in a number of biological processes. Precise regulation of their enzymatic activity is required for normal physiological function. Nedd4-like E3 ligases exist in an inactive form resulting from intramolecular interactions of their catalytic HECT domain with their WW domains. We identified the low-density-lipoprotein receptor class A domain containing 3 (LRAD3), a member of the LDL receptor family, as a potent activator of Itch and Nedd4 as evidenced by their increased auto-ubiquitination when bound to LRAD3. LRAD3 contains two PPxY motifs within its intracellular domain, both of which can bind to the WW domains on Itch and other Nedd4 family members with high affinity. Mutational analysis revealed that binding of Itch to the terminal LRAD3 PPxY motif is required to promote its auto-ubiquitination. We also determined that association of Itch and Nedd4 with LRAD3 leads to increased auto-ubiquitination and subsequent degradation through proteasome-mediated processes. Our findings reveal that LRAD3 is a component of pathways that function effectively to modulate Itch and Nedd4 auto-ubiquitination and levels. The identification of potential ligands for LRAD3 that may modulate LRAD3-induced activation of Itch and Nedd4 is likely to identify additional novel substrates and cellular functions for these important E3 ligases.

Novel approach for generation of low calcium reagents for investigations of heavy metal effects on calcium signaling.

INTRODUCTION: Lead exposure can cause learning disabilities, memory loss and severe damage to the nervous system. However, the exact mechanism by which lead causes learning disabilities is not fully understood. The effects of lead on calcium-regulated signaling pathways are difficult to study biochemically; with the traditional method of controlling the free calcium concentration with EGTA, the exact concentrations of free lead and calcium ions in solution are interdependent and prone to error because EGTA also buffers lead. METHODS AND RESULTS: In our approach, we first reduced the free calcium concentration in the solution using calcium-binding resins before adding lead to buffers. The solution was sequentially treated with Chelex-100 ion exchange resin, followed by immobilized BAPTA resin. The final concentration of free calcium in the solution was measured with Fluo-3 indicator. Our protocol successfully produced buffers with free calcium levels below 15 nM, which is substantially below threshold for activation of calcium-dependent enzymes in signaling pathways (which is typically a few hundred nanomolar calcium, when determined in vitro). CONCLUSION: This method provides an improved approach to study the effect of heavy metals on calcium-stimulated signaling pathways.

LRAD3, a novel low-density lipoprotein receptor family member that modulates amyloid precursor protein trafficking.

We have identified a novel low-density lipoprotein (LDL) receptor family member, termed LDL receptor class A domain containing 3 (LRAD3), which is expressed in neurons. The LRAD3 gene encodes an ∼50 kDa type I transmembrane receptor with an ectodomain containing three LDLa repeats, a transmembrane domain, and a cytoplasmic domain containing a conserved dileucine internalization motif and two polyproline motifs with potential to interact with WW-domain-containing proteins. Immunohistochemical analysis of mouse brain reveals LRAD3 expression in the cortex and hippocampus. In the mouse hippocampal-derived cell line HT22, LRAD3 partially colocalizes with amyloid precursor protein (APP) and interacts with APP as revealed by coimmunoprecipitation experiments. To identify the portion of APP that interacts with LRAD3, we used solid-phase binding assays that demonstrated that LRAD3 failed to bind to a soluble APP fragment (sAPPα) released after α-secretase cleavage. In contrast, C99, the ß-secretase product that remains cell associated, coprecipitated with LRAD3, confirming that regions within this portion of APP are important for associating with LRAD3. The association of LRAD3 with APP increases the amyloidogenic pathway of APP processing, resulting in a decrease in sAPPα production and increased Aß peptide production. Pulse-chase experiments confirm that LRAD3 expression significantly decreases the cellular half-life of mature APP. These results reveal that LRAD3 influences APP processing and raises the possibility that LRAD3 alters APP function in neurons, including its downstream signaling.