Sammut, Michele et al. (2015) International Worm Meeting "The Mystery Cells of the Male: a novel pair of head interneurons required for sex differences in learning."

Barrios, Arantza, Emmons, Scott W., Cook, Steven J., Felton, Terry, Nguyen, Ken, Hall, David H., Sammut, Michele, Poole, Richard J.

[International Worm Meeting, 2015]

We have discovered that our founding fathers overlooked a bilateral pair of male-specific head interneurons, revealing that the total number of male neurons is 385, not 383. We have termed these neurons the Mystery Cells of the Male (MCMs). Matching the level of characterisation performed by Sulston, White, et al. for all other C. elegans neurons, we have established their identity, lineage origin, connectivity and function. Through reporter gene analysis, and ultrastructural reconstruction, we find that the MCMs are fully differentiated neurons. The cell bodies of the MCMs are located dorsoanterior to the pharyngeal metacorpus, a region devoid of neuronal cell bodies in the hermaphrodite, and send projections posteriorly into the nerve ring and along the ventral cord. The MCMs express neuronal gene batteries for both electrical and chemical communication. They are born at the early L4 larval stage from a male-specific asymmetric division of the glial amphid socket (AMso) cells. This is the first example of glia acting as neural progenitors in an invertebrate and parallels vertebrate neurogenesis. We find that the male AMso cells are fully differentiated, just like the hermaphrodite AMso cells, before they re-enter the cell cycle. This male-specific division results in self-renewal of the AMso, which does not retract its projection during division, and a cell that transdifferentiates into the MCM neurons. Reconstruction of the MCM synaptic connectivity through serial reconstruction of electron micrographs reveals a circuit where these neurons create disynaptic pathways in triplet motifs connecting EF (male-specific), AVF, and RIF to AVB. This suggests that the MCMs may incorporate male-specific sensory inputs, such as mate pheromones, into higher-order processing circuits that control chemotactic behaviours. Consistent with this we find that the MCMs are specifically required for sexual conditioning, a mate-experience dependent plasticity in chemotactic responses that trumps the effect of starvation [1].1. Sakai, N., Iwata, R., Yokoi, S., Butcher, R. A., Clardy, J., Tomioka, M., and Iino, Y. (2013). A sexually conditioned switch of chemosensory behavior in C. elegans. PLoS ONE 8, e68676.

Rook, Vicky et al. (2021) International Worm Meeting "Sexually dimorphic glia-neuron reprogramming in Caernorhabditis elegans."

Marti I Sabari , Albert, Rook, Vicky, Sammut , Michele, Bonnington, Rachel, Poole, Richard, Lloret-Fernandez , Carla

[International Worm Meeting, 2021]

How fully differentiated cells can retain their neurogenic potential remains a central question in developmental biology. Previously, we described a glia-to-neuron cell fate switch during sexual maturation in the nervous system of male Caenorhabditis elegans; whereby stably differentiated amphid socket (AMso) glial cells in the head undergo asymmetric cell division to self-renew and give rise to the MCM (Mystery Cells of the Male) neurons1. Using forward genetic screening and candidate gene approaches we have identified a particularly exciting class of mutants (Class III) in which the AMso divides but fails to adopt MCM neuronal identity, instead retaining glial marker expression. We also find that mutations in the zinc finger transcription factor lin-48, an ortholog of vertebrate OVO1 and in the proneural bHLH transcription factor hlh-14, a homolog of vertebrate ASCL1 generate a Class III phenotype. In addition, we have isolated two novel Class III mutants; nom-7 and nom-9 and we are currently performing mapping-by-sequencing to identify the causal locus and will present our most recent findings here. Our analysis of Class III genes will help us to elucidate the molecular mechanisms regulating the plasticity underlying this sexually dimorphic AMso-to-MCM transdifferentiation event. 1Sammut, M. et al. Glia-derived neurons are required for sex-specific learning in C. elegans. Nature 526, 385-390, doi:10.1038/nature15700 (2015).

Molina-Garcia, L. et al. (2017) International Worm Meeting "Sexy learning in C. elegans."

Lin, L., Molina-Garcia, L., Rashid, S., Barrios, A.

[International Worm Meeting, 2017]

We learn from experience. When an animal repeatedly encounters a signal coupled with either a punishment or a reward, it eventually learns to expect both to occur together in a process called associative learning. A central goal in neuroscience is to understand how neural circuits integrate conflicting (rewarding and aversive) experiences that need to be behaviourally resolved during learning. To shed light into this process at the molecular and cellular level, we are dissecting a neural circuit for associative learning in the C. elegans male. Previously, the Iino lab (Sakai et al., 2013) and our lab (Sammut et al., 2015) have shown that C. elegans males undergo sexual conditioning, a form of associative learning by which a rewarding experience with mates overrides the behavioural consequences of an aversive association with starvation. Sexual conditioning leads to a switch in behavioural responses to an environmental stimulus from avoidance to attraction. We have also identified the MCM interneurons and PDF neuromodulation as regulators of the sexually conditioned behavioural switch (Sammut et al.2015). These studies showed conditioned responses to salt. Here we demonstrate that other stimuli such as benzaldehyde or diacetyl can also be sexually conditioned. In addition, we show that mutants without MCMs and mutants in the neuropeptide pdf-1, which is expressed in the MCMs, fail to undergo sexual conditioning also to odorants. These results indicate that sexual conditioning is a general, broadly employed strategy of the male to effectively search for and locate mates during navigation. Furthermore, the MCM neurons and PDF neuromodulation are common mechanisms underlying sexual conditioning to distinct stimuli. We are now interrogating the circuit for chemotaxis learning to identify the neurons within it that are modulated by the MCMs and PDF neuropeptide.

Molina-Garcia, Laura et al. (2021) International Worm Meeting "PDF-1 modulation of aversion and reward during associative learning"

Barrios, Arantza, Lin, Lucy, Colinas-Fischer, Susana, Molina-Garcia, Laura

[International Worm Meeting, 2021]

A central goal in neuroscience is to understand how neural circuits integrate conflicting (rewarding and aversive) experiences that need to be behaviourally resolved during learning. To shed light into the molecular and cellular mechanisms underlying this process we are dissecting the neuronal circuit that regulates sexual conditioning in C. elegans. Previously, the Iino lab (Sakai et al., 2013) and us (Sammut et al., 2015) have shown that C. elegans males undergo sexual conditioning, a form of associative learning by which a rewarding experience with mates overrides the behavioural consequences of an aversive association with starvation. Thus, sexual conditioning leads to a switch in behavioural responses to an environmental stimulus from avoidance to attraction. Our lab also identified the MCM interneurons and PDF-1 neuromodulation as the cellular and molecular regulators of the sexually conditioned behavioural switch (Sammut et al., 2015). These studies showed conditioned responses to salt. Here we show that other stimuli such as benzaldehyde can also be sexually conditioned and this is also regulated by the MCMs and PDF-1. We find a dual role for PDF-1 in the regulation of aversive learning and sexual conditioning in C. elegans. By using a Cre-Lox intersectional strategy we found that PDF signaling encodes both aversion and sexual conditioning by modulating two partially distinct circuits. Within these circuits, we have identified the interneurons RIA, AIY and RIM as target cells receiving PDF-1 neuromodulation to drive sexual conditioning. Expression of the PDF-1 receptor (pdfr-1) only in these neurons is sufficient to rescue attraction during sexual conditioning, but not repulsion during aversive learning. Furthermore, by removing PDF-1 in specific cells, while keeping the endogenous levels of expression in the rest of the circuit, we have identified the MCMs and AVB neurons, as a source of PDF-1 during sexual conditioning. Importantly, PDF-1 release from the MCMs and AVBs is not required during aversive learning. Thus, highlighting the importance of source specificity versus overall neuropeptide levels during fine tune modulation of specific behaviors. Currently, we are measuring neuronal activity within the chemotaxis circuit to further understand how sensory information is represented after conditioning and PDF neuromodulation to switch odour preferences.

Emmons, S et al. (2015) International Worm Meeting "Whole-animal C. elegans connectomes."

Emmons, S, Wang, Y, Nguyen, K, Jarrell, T, Cook, S, Yakovlev, M, Hall, D

[International Worm Meeting, 2015]

We have assembled for the first time complete wiring diagrams of the entire nervous systems of C. elegans adults of both sexes. For the hermaphrodite, we reanalyzed the electron micrographs that served as the basis for the 1986 publication "The Mind of a Worm" (White et al, 1986). For the male, we combined the posterior connectivity we published previously (Jarrell et al, 2012), obtained by analyzing Cambridge electron micrographs (Sulston et al, 1970), with data from our own series through a male head. For the pharynx we reanalyzed the micrographs analyzed previously by Donna Albertson (Albertson and Thomson, 1976). For some neurons, we added data from new electron micrographs of legacy grids. Notably, we added connectivity of SAB motorneurons to body wall muscles in the head (muscles 1-7); these neurons contribute 37% of the total input to these muscles. Finally, significant gaps still remained: to our knowledge, a region posterior of the vulva (posterior of the N2U series, anterior of the male N2Y series) has never been examined by electron microscopy. To fill in these areas, we added connections to complete the chains of motor neurons and muscles by assuming a regular structure. Connectivity matrices, neuron maps, and synapse lists are available on our website: WormWiring.org. Analysis of the complete, quantitative datasets by graph layout algorithms yields biologically meaningful displays. When an algorithm is applied that utilizes the "spring-electric" approach, in which nodes repel each other uniformly and attract according to the strength of their connectivity (Allegro Layout, allegroviva.com), neurons of similar or related function are grouped together. At the next higher level, clear modules are separated. When the arrangement is graphically illustrated using the display tools of Cytoscape, the functional pathways and hierarchical arrangement of the entire worm neuromuscular system are revealed-the layout closely matches the worm's anatomy. The overall arrangement is the same in both sexes, with the addition in the hermaphrodite of the vulva muscles and circuits and to the male "tail" of the enormous copulatory circuits. Along with the sensory pathways for head contact and for odorants and chemicals, pathways leading from pheromone sensing neurons reveal a sexual circuit in the head, elements of which (AVF, PVQ, and RIM interneurons) are shared by both sexes, along with, in the hermaphrodite, the HSN neurons, and in the male, the CEM sensory neurons and the EF and MCM interneurons (for the new MCM interneurons, see the Abstract by Sammut et al).