Neuromodulatory pathways in learning and memory: Lessons from invertebrates.

In an ever-changing environment, animals have to continuously adapt their behaviour. The ability to learn from experience is crucial for animals to increase their chances of survival. It is therefore not surprising that learning and memory evolved early in evolution and are mediated by conserved molecular mechanisms. A broad range of neuromodulators, in particular monoamines and neuropeptides, have been found to influence learning and memory, although our knowledge on their modulatory functions in learning circuits remains fragmentary. Many neuromodulatory systems are evolutionarily ancient and well-conserved between vertebrates and invertebrates. Here, we highlight general principles and mechanistic insights concerning the actions of monoamines and neuropeptides in learning circuits that have emerged from invertebrate studies. Diverse neuromodulators have been shown to influence learning and memory in invertebrates, which can have divergent or convergent actions at different spatiotemporal scales. In addition, neuromodulators can regulate learning dependent on internal and external states, such as food and social context. The strong conservation of neuromodulatory systems, the extensive toolkit and the compact learning circuits in invertebrate models make these powerful systems to further deepen our understanding of neuromodulatory pathways involved in learning and memory.

NPY/NPF-Related Neuropeptide FLP-34 Signals from Serotonergic Neurons to Modulate Aversive Olfactory Learning in Caenorhabditis elegans.

Aversive learning is fundamental for animals to increase chances of survival. In addition to classical neurotransmitters, neuropeptides have emerged to modulate such complex behaviors. Among them, neuropeptide Y (NPY) is well known to promote aversive memory acquisition in mammals. Here we identify an NPY/neuropeptide F (NPF)-related neuropeptide system in Caenorhabditis elegans and show that this FLP-34/NPR-11 system is required for learning negative associations, a process that is reminiscent of NPY signaling in mammals. The Caenorhabditis elegans NPY/NPF ortholog FLP-34 displays conserved structural hallmarks of bilaterian-wide NPY/NPF neuropeptides. We show that it is required for aversive olfactory learning after pairing diacetyl with the absence of food, but not for appetitive olfactory learning in response to butanone. To mediate diacetyl learning and thus integrate the aversive food context with the diacetyl odor, FLP-34 is released from serotonergic neurons and signals through its evolutionarily conserved NPY/NPF GPCR, NPR-11, in downstream AIA interneurons. NPR-11 activation in the AIA integration center results in avoidance of a previously attractive stimulus. This study opens perspectives for a deeper understanding of stress conditions in which aversive learning results in excessive avoidance.SIGNIFICANCE STATEMENT Aversive learning evolved early in evolution to promote avoidance of dangerous and stressful situations. In addition to classical neurotransmitters, neuropeptides are emerging as modulators of complex behaviors, including learning and memory. Here, we identified the evolutionary ortholog of neuropeptide Y/neuropeptide F in the nematode Caenorhabditis elegans, and we discovered that it is required for olfactory aversive learning. In addition, we elucidated the neural circuit underlying this avoidance behavior, and we discovered a novel coordinated action of Caenorhabditis elegans neuropeptide Y/neuropeptide F and serotonin that could aid in our understanding of the molecular mechanisms underlying stress disorders in which excessive avoidance results in maladaptive behaviors.

Neuromedin U signaling regulates retrieval of learned salt avoidance in a C. elegans gustatory circuit.

Learning and memory are regulated by neuromodulatory pathways, but the contribution and temporal requirement of most neuromodulators in a learning circuit are unknown. Here we identify the evolutionarily conserved neuromedin U (NMU) neuropeptide family as a regulator of C. elegans gustatory aversive learning. The NMU homolog CAPA-1 and its receptor NMUR-1 are required for the retrieval of learned salt avoidance. Gustatory aversive learning requires the release of CAPA-1 neuropeptides from sensory ASG neurons that respond to salt stimuli in an experience-dependent manner. Optogenetic silencing of CAPA-1 neurons blocks the expression, but not the acquisition, of learned salt avoidance. CAPA-1 signals through NMUR-1 in AFD sensory neurons to modulate two navigational strategies for salt chemotaxis. Aversive conditioning thus recruits NMU signaling to modulate locomotor programs for expressing learned avoidance behavior. Because NMU signaling is conserved across bilaterian animals, our findings incite further research into its function in other learning circuits.

Myoinhibitory peptide signaling modulates aversive gustatory learning in Caenorhabditis elegans.

Aversive learning and memories are crucial for animals to avoid previously encountered stressful stimuli and thereby increase their chance of survival. Neuropeptides are essential signaling molecules in the brain and are emerging as important modulators of learned behaviors, but their precise role is not well understood. Here, we show that neuropeptides of the evolutionarily conserved MyoInhibitory Peptide (MIP)-family modify salt chemotaxis behavior in Caenorhabditis elegans according to previous experience. MIP signaling, through activation of the G protein-coupled receptor SPRR-2, is required for short-term gustatory plasticity. In addition, MIP/SPRR-2 neuropeptide-receptor signaling mediates another type of aversive gustatory learning called salt avoidance learning that depends on de novo transcription, translation and the CREB transcription factor, all hallmarks of long-term memory. MIP/SPRR-2 signaling mediates salt avoidance learning in parallel with insulin signaling. These findings lay a foundation to investigate the suggested orphan MIP receptor orthologs in deuterostomians, including human GPR139 and GPR142.

Evolutionarily conserved TRH neuropeptide pathway regulates growth in Caenorhabditis elegans.

In vertebrates thyrotropin-releasing hormone (TRH) is a highly conserved neuropeptide that exerts the hormonal control of thyroid-stimulating hormone (TSH) levels as well as neuromodulatory functions. However, a functional equivalent in protostomian animals remains unknown, although TRH receptors are conserved in proto- and deuterostomians. Here we identify a TRH-like neuropeptide precursor in Caenorhabditis elegans that belongs to a bilaterian family of TRH precursors. Using CRISPR/Cas9 and RNAi reverse genetics, we show that TRH-like neuropeptides, through the activation of their receptor TRHR-1, promote growth in Celegans TRH-like peptides from pharyngeal motor neurons are required for normal body size, and knockdown of their receptor in pharyngeal muscle cells reduces growth. Mutants deficient for TRH signaling have no defects in pharyngeal pumping or isthmus peristalsis rates, but their growth defect depends on the bacterial diet. In addition to the decrease in growth, trh-1 mutants have a reduced number of offspring. Our study suggests that TRH is an evolutionarily ancient neuropeptide, having its origin before the divergence of protostomes and deuterostomes, and may ancestrally have been involved in the control of postembryonic growth and reproduction.

Characterization of a neuropeptide F receptor in the tsetse fly, Glossina morsitans morsitans.

Neuropeptides related to mammalian neuropeptide Y (NPY) and insect neuropeptide F (NPF) are conserved throughout Metazoa and intimately involved in a wide range of biological processes. In insects NPF is involved in regulating feeding, learning, stress and reproductive behavior. Here we identified and characterized an NPF receptor of the tsetse fly, Glossina morsitans morsitans, the sole transmitter of Trypanosoma parasites causing sleeping sickness. We isolated cDNA sequences encoding tsetse NPF (Glomo-NPF) and its receptor (Glomo-NPFR), and examined their spatial and temporal expression patterns using quantitative PCR. In tsetse flies, npfr transcripts are expressed throughout development and most abundantly in the central nervous system, whereas low expression is found in the flight muscles and posterior midgut. Expression of npf, by contrast, shows low transcript levels during development but is strongly expressed in the posterior midgut and brain of adult flies. Expression of Glomo-npf and its receptor in the brain and digestive system suggests that NPF may have conserved neuromodulatory or hormonal functions in tsetse flies, such as in the regulation of feeding behavior. Cell-based activity studies of the Glomo-NPFR showed that Glomo-NPF activates the receptor up to nanomolar concentrations. The molecular data of Glomo-NPF and Glomo-NPFR paves the way for further investigation of its functions in tsetse flies.

Molecular characterization of a short neuropeptide F signaling system in the tsetse fly, Glossina morsitans morsitans.

Neuropeptides of the short neuropeptide F (sNPF) family are widespread among arthropods and found in every sequenced insect genome so far. Functional studies have mainly focused on the regulatory role of sNPF in feeding behavior, although this neuropeptide family has pleiotropic effects including in the control of locomotion, osmotic homeostasis, sleep, learning and memory. Here, we set out to characterize and determine possible roles of sNPF signaling in the haematophagous tsetse fly Glossina morsitans morsitans, a vector of African Trypanosoma parasites causing human and animal African trypanosomiasis. We cloned the G. m. morsitans cDNA sequences of an sNPF-like receptor (Glomo-sNPFR) and precursor protein encoding four Glomo-sNPF neuropeptides. All four Glomo-sNPF peptides concentration-dependently activated Glomo-sNPFR in a cell-based calcium mobilization assay, with EC50 values in the nanomolar range. Gene expression profiles in adult female tsetse flies indicate that the Glomo-sNPF system is mainly restricted to the nervous system. Glomo-snpfr transcripts were also detected in the hindgut of adult females. In contrast to the Drosophila sNPF system, tsetse larvae lack expression of Glomo-snpf and Glomo-snpfr genes. While Glomo-snpf transcript levels are upregulated in pupae, the onset of Glomo-snpfr expression is delayed to adulthood. Expression profiles in adult tissues are similar to those in other insects suggesting that the tsetse sNPF system may have similar functions such as a regulatory role in feeding behavior, together with a possible involvement of sNPFR signaling in osmotic homeostasis. Our molecular data will enable further investigations into the functions of sNPF signaling in tsetse flies.

Corrigendum: the FMRFamide-like peptide family in nematodes.

[This corrects the article on p. 90 in vol. 5, PMID: 24982652.].

Characterization of G protein-coupled receptors by a fluorescence-based calcium mobilization assay.

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα16 construct is co-transfected. Following ligand binding, activation of the Gα16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The FMRFamide-Like Peptide Family in Nematodes.

In the three decades since the FMRFamide peptide was isolated from the mollusk Macrocallista nimbosa, structurally similar peptides sharing a C-terminal RFamide motif have been identified across the animal kingdom. FMRFamide-like peptides (FLPs) represent the largest known family of neuropeptides in invertebrates. In the phylum Nematoda, at least 32 flp-genes are classified, making the FLP system of nematodes unusually complex. The diversity of the nematode FLP complement is most extensively mapped in Caenorhabditis elegans, where over 70 FLPs have been predicted. FLPs have shown to be expressed in the majority of the 302 C. elegans neurons including interneurons, sensory neurons, and motor neurons. The vast expression of FLPs is reflected in the broad functional repertoire of nematode FLP signaling, including neuroendocrine and neuromodulatory effects on locomotory activity, reproduction, feeding, and behavior. In contrast to the many identified nematode FLPs, only few peptides have been assigned a receptor and there is the need to clarify the pathway components and working mechanisms of the FLP signaling network. Here, we review the diversity, distribution, and functions of FLPs in nematodes.