Metamorphosis of memory circuits in Drosophila reveals a strategy for evolving a larval brain.

Mushroom bodies (MB) of adult Drosophila have a core of thousands of Kenyon neurons; axons of the early-born g class form a medial lobe and those from later-born α'ß' and αß classes form both medial and vertical lobes. The larva, however, hatches with only γ neurons and forms a vertical lobe 'facsimile' using larval-specific axon branches from its γ neurons. MB input (MBINs) and output (MBONs) neurons divide the Kenyon neuron lobes into discrete computational compartments. The larva has 10 such compartments while the adult has 16. We determined the fates of 28 of the 32 MBONs and MBINs that define the 10 larval compartments. Seven compartments are subsequently incorporated into the adult MB; four of their MBINs die, while 12 MBINs/MBONs remodel to function in adult compartments. The remaining three compartments are larval specific. At metamorphosis their MBIN/MBONs trans-differentiate, leaving the MB for other adult brain circuits. The adult vertical lobes are made de novo using MBONs/MBINs recruited from pools of adult-specific neurons. The combination of cell death, compartment shifting, trans-differentiation, and recruitment of new neurons result in no larval MBIN-MBON connections being maintained through metamorphosis. At this simple level, then, we find no anatomical substrate for a memory trace persisting from larva to adult. The adult phenotype of the trans-differentiating neurons represents their evolutionarily ancestral phenotype while their larval phenotype is a derived adaptation for the larval stage. These cells arise primarily within lineages that also produce permanent MBINs and MBONs, suggesting that larval specifying factors may allow information related to birth-order or sibling identity to be interpreted in a modified manner in the larva to allow these neurons to acquire larval phenotypic modifications. The loss of such factors at metamorphosis then allows these neurons to revert to their ancestral functions in the adult.

Hormone-controlled changes in the differentiation state of post-mitotic neurons.

While we think of neurons as having a fixed identity, many show spectacular plasticity.1-10 Metamorphosis drives massive changes in the fly brain;11,12 neurons that persist into adulthood often change in response to the steroid hormone ecdysone.13,14 Besides driving remodeling,11-14 ecdysone signaling can also alter the differentiation status of neurons.7,15 The three sequentially born subtypes of mushroom body (MB) Kenyon cells (γ, followed by α'/ß', and finally α/ß)16 serve as a model of temporal fating.17-21 γ neurons are also used as a model of remodeling during metamorphosis. As γ neurons are the only functional Kenyon cells in the larval brain, they serve the function of all three adult subtypes. Correspondingly, larval γ neurons have a similar morphology to α'/ß' and α/ß neurons-their axons project dorsally and medially. During metamorphosis, γ neurons remodel to form a single medial projection. Both temporal fate changes and defects in remodeling therefore alter γ-neuron morphology in similar ways. Mamo, a broad-complex, tramtrack, and bric-à-brac/poxvirus and zinc finger (BTB/POZ) transcription factor critical for temporal specification of α'/ß' neurons,18,19 was recently described as essential for γ remodeling.22 In a previous study, we noticed a change in the number of adult Kenyon cells expressing γ-specific markers when mamo was manipulated.18 These data implied a role for Mamo in γ-neuron fate specification, yet mamo is not expressed in γ neurons until pupariation,18,22 well past γ specification. This indicates that mamo has a later role in ensuring that γ neurons express the correct Kenyon cell subtype-specific genes in the adult brain.

General anesthetics activate a nociceptive ion channel to enhance pain and inflammation.

General anesthetics (GAs) have transformed surgery through their actions to depress the central nervous system and blunt the perception of surgical insults. Counterintuitively, many of these agents activate peripheral nociceptive neurons. However, the underlying mechanisms and significance of these effects have not been explored. Here, we show that clinical concentrations of noxious i.v. and inhalation GAs excite sensory neurons by selectively activating TRPA1, a key ion channel in the pain pathway. Further, these GAs induce pain-related responses in mice that are abolished in TRPA1-null animals. Significantly, TRPA1-dependent neurogenic inflammation is greater in mice anesthetized with pungent compared with nonpungent anesthetics. Thus, our results show that TRPA1 is essential for sensing noxious GAs. The pronociceptive effects of GAs combined with surgical tissue damage could lead to a paradoxical increase in postoperative pain and inflammation.

Conservation and divergence of related neuronal lineages in the Drosophila central brain.

Wiring a complex brain requires many neurons with intricate cell specificity, generated by a limited number of neural stem cells. Drosophila central brain lineages are a predetermined series of neurons, born in a specific order. To understand how lineage identity translates to neuron morphology, we mapped 18 Drosophila central brain lineages. While we found large aggregate differences between lineages, we also discovered shared patterns of morphological diversification. Lineage identity plus Notch-mediated sister fate govern primary neuron trajectories, whereas temporal fate diversifies terminal elaborations. Further, morphological neuron types may arise repeatedly, interspersed with other types. Despite the complexity, related lineages produce similar neuron types in comparable temporal patterns. Different stem cells even yield two identical series of dopaminergic neuron types, but with unrelated sister neurons. Together, these phenomena suggest that straightforward rules drive incredible neuronal complexity, and that large changes in morphology can result from relatively simple fating mechanisms.

Mamo decodes hierarchical temporal gradients into terminal neuronal fate.

Temporal patterning is a seminal method of expanding neuronal diversity. Here we unravel a mechanism decoding neural stem cell temporal gene expression and transforming it into discrete neuronal fates. This mechanism is characterized by hierarchical gene expression. First, Drosophila neuroblasts express opposing temporal gradients of RNA-binding proteins, Imp and Syp. These proteins promote or inhibit chinmo translation, yielding a descending neuronal gradient. Together, first and second-layer temporal factors define a temporal expression window of BTB-zinc finger nuclear protein, Mamo. The precise temporal induction of Mamo is achieved via both transcriptional and post-transcriptional regulation. Finally, Mamo is essential for the temporally defined, terminal identity of α'/ß' mushroom body neurons and identity maintenance. We describe a straightforward paradigm of temporal fate specification where diverse neuronal fates are defined via integrating multiple layers of gene regulation. The neurodevelopmental roles of orthologous/related mammalian genes suggest a fundamental conservation of this mechanism in brain development.

Zebrafish yolk lipid processing: a tractable tool for the study of vertebrate lipid transport and metabolism.

Dyslipidemias are a major cause of morbidity and mortality in the world, particularly in developed nations. Investigating lipid and lipoprotein metabolism in experimentally tractable animal models is a crucial step towards understanding and treating human dyslipidemias. The zebrafish, a well-established embryological model, is emerging as a notable system for studies of lipid metabolism. Here, we describe the value of the lecithotrophic, or yolk-metabolizing, stages of the zebrafish as a model for studying lipid metabolism and lipoprotein transport. We demonstrate methods to assay yolk lipid metabolism in embryonic and larval zebrafish. Injection of labeled fatty acids into the zebrafish yolk promotes efficient uptake into the circulation and rapid metabolism. Using a genetic model for abetalipoproteinemia, we show that the uptake of labeled fatty acids into the circulation is dependent on lipoprotein production. Furthermore, we examine the metabolic fate of exogenously delivered fatty acids by assaying their incorporation into complex lipids. Moreover, we demonstrate that this technique is amenable to genetic and pharmacologic studies.

TRPV1 is a novel target for omega-3 polyunsaturated fatty acids.

Omega-3 (n-3) fatty acids are essential for proper neuronal function, and they possess prominent analgesic properties, yet their underlying signalling mechanisms are unclear. Here we show that n-3 fatty acids interact directly with TRPV1, an ion channel expressed in nociceptive neurones and brain. These fatty acids activate TRPV1 in a phosphorylation-dependent manner, enhance responses to extracellular protons, and displace binding of the ultrapotent TRPV1 ligand [3H]resiniferatoxin. In contrast to their agonistic properties, n-3 fatty acids competitively inhibit the responses of vanilloid agonists. These actions occur in mammalian cells in the physiological concentration range of 1-10 mum. Significantly, docosahexaenoic acid exhibits the greatest efficacy as an agonist, whereas eicosapentaenoic acid and linolenic acid are markedly more effective inhibitors. Similarly, eicosapentaenoic acid but not docosahexaenoic acid profoundly reduces capsaicin-evoked pain-related behaviour in mice. These effects are independent of alterations in membrane elasticity because the micelle-forming detergent Triton X-100 only minimally affects TRPV1 properties. Thus, n-3 fatty acids differentially regulate TRPV1 and this form of signalling may contribute to their biological effects. Further, these results suggest that dietary supplementation with selective n-3 fatty acids would be most beneficial for the treatment of pain.