Restructuring of an asymmetric neural circuit during associative learning

Leo T.H. Tang, Garrett A. Lee, Steven J. Cook, Jacquelin Ho, Cassandra C. Potter, Hannes E. Bülow

Preprint posted on 13 January 2023

The only constant is change – even in “hard-wired” C. elegans. Rewiring of an asymmetric neural circuit leads to associative salt-learning.

Selected by Chee Kiang Ewe


In 1977, John Sulston and Bob Horvitz traced the cell lineage of C. elegans larvae and found that C. elegans development is highly robust; hermaphrodites contain 1090 somatic cells, 131 of which undergo programmed cell death, leaving invariant 959 cells (Sulston et al., 1983; Sulston & Horvitz, 1977). Around the same time, John White reconstructed the connectome, which consists of 302 stereotyped neurons, using electron microscopy (White et al., 1986). While these classical studies imply that C. elegans development, just as its nervous system, is hardwired, a recent preprint demonstrates that synaptic connectivity may undergo significant restructuring during associative learning. The authors of this preprint could show that salt conditioning alters the architecture of the salt sensing and learning circuit, which is controlled by the conserved insulin signaling (IIS) pathway. This study therefore provides an important insight into the molecular basis for the specification of asymmetric brain functionality.

Key findings:

  • Salt exposure leads to rewiring of synaptic connectivity and associative learning in elegans.

As shown previously (Kunitomo et al., 2013), the authors reported that (naïve) worms grown in low salt (33 mM) conditions prefer a low salt environment over a high salt environment in a chemotaxis assay. In contrast, worms grown in high salt (100 mM) conditions for 12 hours tended to chemotax towards agar with a high salt content. C. elegans detects salt using a pair of ASE sensory neurons (left: ASEL; right: ASER). Using a reporter for the presynaptic active zone and iBLINC technology, the authors noted that naïve worms showed a left bias, while conditioned worms showed a right bias in the ASE connection with their postsynaptic partners AWCL/R. The switch in left/right bias in ASE>AWC connection appears to drive differential salt preference, demonstrating that synaptic “hardwiring” is indeed plastic and may change with experience.

  • ASE cell fate in part determines asymmetric connectivity.

Transcriptional specification drives left/right asymmetry in both ASE and AWC neurons (Hobert, 2014). Intriguingly, the authors found that symmetrizing ASE, but not AWC, altered ASE>AWC connectivity. Left biased ASE>AWC connectivity was observed when both ASE adopted an ASER fate, as in naïve wildtype; however, a right bias was observed when both ASE adopted an ASEL fate, indicating that while ASE fate may influence left/right asymmetric connectivity, there are other unknown factors at play here since asymmetry persists even when ASE neurons are symmetrized.

  • The IIS pathway regulates synaptic connectivity.

It was previously found that the IIS pathway is important for salt sensing and learning (Tomioka et al., 2006). Indeed, the authors showed that knocking out the gene encoding the insulin receptor daf-2 or the downstream PI3 kinase age-1, abrogated asymmetry in ASE>AWC connectivity. Using the Cre-Lox system to achieve cell-specific knockouts, the authors showed that DAF-2 plays a role in establishing asymmetric connectivity in ASE, but not in AWC. Interestingly, removing daf-2 in ASEL in naïve worms led to a reduced connectivity on the left side and a more symmetric pattern. Nevertheless, salt conditioned mutant worms still showed a right-side bias. In contrast, eliminating daf-2 in ASER did not affect connectivity in naïve worms, but conditioned animals showed symmetrized ASE>AWC connections. These results indicate that the IIS pathway promotes synaptic formation and that DAF-2 is asymmetrically activated. Indeed, by examining the localization of DAF-16/FOXO (IIS effector), the authors could show that the IIS pathway is more active in ASEL in naïve worms and in ASER in conditioned worms, thereby restructuring the synaptic connectivity during associative learning.

Next, the authors examined the involvement of insulin-like peptide in ASE>AWC connectivity and found, surprisingly, that worms lacking ins-6, unlike daf-2(-) mutants, showed a right bias and preferred high salt agar. This indicates that while INS-6 is important in regulating ASE>AWC connectivity, the outcome of DAF-2 activation is likely modulated by multiple insulin-like peptides. Interestingly, the authors found that INS-6 was expressed in two pairs of ASI and ASJ sensory neurons. While the expression of ins-6 is symmetric in ASI with or without salt conditioning, it is stronger in ASJL than in ASJR in naïve animals; the reverse is true in conditioned animals, mirroring ASE>AWC connectivity. Intriguingly, eliminating ins-6 in ASJ alone symmetrized ASE>AWC connectivity, whereas removing ins-6 in both ASJ and ASI led to a right bias, recapitulating the ins-6(-) mutant’s phenotype. Finally, the authors knocked out ins-6 specifically in ASJL, but not in ASJR, and observed a right bias in ASE>AWC connectivity. These results indicate that both the expression and the source (ASJ vs. ASI) of INS-6 are important in modulating ASE>AWC asymmetry (Figure 1).

Figure 1: INS-6 and the IIS control the left/right asymmetry of ASE>AWC connectivity (created with

What I liked about this preprint:

Tang and colleagues beautifully show how asymmetrical activation of IIS leads to associative learning in C. elegans. The experiments are thorough, and I am especially impressed by the clever use of the Cre-Lox system to generate cell-specific knockouts. As the authors have pointed out, the expression of insulin-like peptide in the nervous system is evolutionary conserved, raising the exciting possibility that IIS may also mediate changes in neuronal network architecture in other organisms.

Questions for the authors:

  • It is very interesting that knocking out ins-6 in ASJ or ASI gives rise to different phenotypes. How do you think ASE distinguishes INS-6 released from ASJ or ASI?
  • Have you tried conditioning the worms at L1 or L2 to see if that affects the establishment of the asymmetric connectivity later in their lives?


Hobert, O. (2014). Development of left/right asymmetry in the Caenorhabditis elegans nervous system: From zygote to postmitotic neuron. Genesis, 52(6), 528–543.

Kunitomo, H., Sato, H., Iwata, R., Satoh, Y., Ohno, H., Yamada, K., & Iino, Y. (2013). Concentration memory-dependent synaptic plasticity of a taste circuit regulates salt concentration chemotaxis in Caenorhabditis elegans. Nature Communications, 4.

Sulston, J. E., & Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Developmental Biology, 56(1), 110–156.

Sulston, J. E., Schierenberg, E., White, J. G., & Thomson, J. N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Developmental Biology, 100(1), 64–119.

Tomioka, M., Adachi, T., Suzuki, H., Kunitomo, H., Schafer, W. R., & Iino, Y. (2006). The insulin/PI 3-kinase pathway regulates salt chemotaxis learning in Caenorhabditis elegans. Neuron, 51(5), 613–625.

White, J. G., Southgate, E., Thomson, J. N., & Brenner, S. (1986). The structure of the nervous system of the nematode Caenorhabditis elegans. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 314(1165), 1–340.



Tags: associative learning, c. elegans, insulin signaling, neurogenetics

Posted on: 30 January 2023


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