Differences between female and male brains exist across the animal kingdom and extend from molecular to anatomical features. Here we show that sexually dimorphic anatomy, gene expression and function in the nervous system can be modulated by past experiences. In the nematode Caenorhabditis elegans, sexual differentiation entails the sex-specific pruning of synaptic connections between neurons that are shared by both sexes, giving rise to sexually dimorphic circuits in adult animals1. We discovered that starvation during juvenile stages is memorized in males to suppress the emergence of sexually dimorphic synaptic connectivity. These circuit changes result in increased chemosensory responsiveness in adult males following juvenile starvation. We find that an octopamine-mediated starvation signal dampens the production of serotonin (5-HT) to convey the memory of starvation. Serotonin production is monitored by a 5-HT1A serotonin receptor homologue that acts cell-autonomously to promote the pruning of sexually dimorphic synaptic connectivity under well-fed conditions. Our studies demonstrate how life history shapes neurotransmitter production, synaptic connectivity and behavioural output in a sexually dimorphic circuit.
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Oren-Suissa, M., Bayer, E. A. & Hobert, O. Sex-specific pruning of neuronal synapses in Caenorhabditis elegans. Nature 533, 206–211 (2016).
Barr, M. M., García, L. R. & Portman, D. S. Sexual dimorphism and sex differences in Caenorhabditis elegans neuronal development and behavior. Genetics 208, 909–935 (2018).
White, J. G., Southgate, E., Thomson, J. N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Phil. Trans. R. Soc. Lond. B 314, 1–340 (1986).
Jarrell, T. A. et al. The connectome of a decision-making neural network. Science 337, 437–444 (2012).
Hilliard, M. A., Bargmann, C. I. & Bazzicalupo, P. C. elegans responds to chemical repellents by integrating sensory inputs from the head and the tail. Curr. Biol. 12, 730–734 (2002).
Lipton, J., Kleemann, G., Ghosh, R., Lints, R. & Emmons, S. W. Mate searching in Caenorhabditis elegans: a genetic model for sex drive in a simple invertebrate. J. Neurosci. 24, 7427–7434 (2004).
Liu, K. S. & Sternberg, P. W. Sensory regulation of male mating behavior in Caenorhabditis elegans. Neuron 14, 79–89 (1995).
Sherlekar, A. L. et al. The C. elegans male exercises directional control during mating through cholinergic regulation of sex-shared command interneurons. PLoS ONE 8, e60597 (2013).
Goldstein, J. L. et al. Surviving starvation: essential role of the ghrelin-growth hormone axis. Cold Spring Harb. Symp. Quant. Biol. 76, 121–127 (2011).
Churgin, M. A., McCloskey, R. J., Peters, E. & Fang-Yen, C. Antagonistic serotonergic and octopaminergic neural circuits mediate food-dependent locomotory behavior in Caenorhabditis elegans. J. Neurosci. 37, 7811–7823 (2017).
Harris, G. et al. The monoaminergic modulation of sensory-mediated aversive responses in Caenorhabditis elegans requires glutamatergic/peptidergic cotransmission. J. Neurosci. 30, 7889–7899 (2010).
Jafari, G., Xie, Y., Kullyev, A., Liang, B. & Sze, J. Y. Regulation of extrasynaptic 5-HT by serotonin reuptake transporter function in 5-HT-absorbing neurons underscores adaptation behavior in Caenorhabditis elegans. J. Neurosci. 31, 8948–8957 (2011).
Alkema, M. J., Hunter-Ensor, M., Ringstad, N. & Horvitz, H. R. Tyramine functions independently of octopamine in the Caenorhabditis elegans nervous system. Neuron 46, 247–260 (2005).
Tao, J., Ma, Y. C., Yang, Z. S., Zou, C. G. & Zhang, K. Q. Octopamine connects nutrient cues to lipid metabolism upon nutrient deprivation. Sci. Adv. 2, e1501372 (2016).
Liang, B., Moussaif, M., Kuan, C. J., Gargus, J. J. & Sze, J. Y. Serotonin targets the DAF-16/FOXO signaling pathway to modulate stress responses. Cell Metab. 4, 429–440 (2006).
Noble, T., Stieglitz, J. & Srinivasan, S. An integrated serotonin and octopamine neuronal circuit directs the release of an endocrine signal to control C. elegans body fat. Cell Metab. 18, 672–684 (2013).
Carre-Pierrat, M. et al. Characterization of the Caenorhabditis elegans G protein-coupled serotonin receptors. Invert. Neurosci. 6, 189–205 (2006).
Harris, G. P. et al. Three distinct amine receptors operating at different levels within the locomotory circuit are each essential for the serotonergic modulation of chemosensation in Caenorhabditis elegans. J. Neurosci. 29, 1446–1456 (2009).
Gürel, G., Gustafson, M. A., Pepper, J. S., Horvitz, H. R. & Koelle, M. R. Receptors and other signaling proteins required for serotonin control of locomotion in Caenorhabditis elegans. Genetics 192, 1359–1371 (2012).
Riad, M. et al. Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain. J. Comp. Neurol. 417, 181–194 (2000).
Lajud, N. & Torner, L. Early life stress and hippocampal neurogenesis in the neonate: sexual dimorphism, long term consequences and possible mediators. Front. Mol. Neurosci. 8, 3 (2015).
Houwing, D. J., Buwalda, B., van der Zee, E. A., de Boer, S. F. & Olivier, J. D. A. The serotonin transporter and early life stress: translational perspectives. Front. Cell. Neurosci. 11, 117 (2017).
van den Hove, D. L. et al. Differential effects of prenatal stress in 5-Htt deficient mice: towards molecular mechanisms of gene × environment interactions. PLoS ONE 6, e22715 (2011).
Zheng, X., Chung, S., Tanabe, T. & Sze, J. Y. Cell-type specific regulation of serotonergic identity by the C. elegans LIM-homeodomain factor LIM-4. Dev. Biol. 286, 618–628 (2005).
Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).
Feinberg, E. H. et al. GFP reconstitution across synaptic partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron 57, 353–363 (2008).
Desbois, M., Cook, S. J., Emmons, S. W. & Bülow, H. E. Directional trans-synaptic labeling of specific neuronal connections in live animals. Genetics 200, 697–705 (2015).
Ji, N. & van Oudenaarden, A. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos. WormBook https://doi.org/10.1895/wormbook.1.153.1 (2012).
Zhang, F. et al. The LIM and POU homeobox genes ttx-3 and unc-86 act as terminal selectors in distinct cholinergic and serotonergic neuron types. Development 141, 422–435 (2014).
We thank Q. Chen for generating transgenic strains, K. Ashrafi for providing DNA constructs, the CGC (supported by the NIH P40 OD010440) for strains and members of the Hobert laboratory, I. Greenwald, and M. Oren-Suissa for comments on the manuscript. This work was supported by the HHMI and NIH (R37NS039996, O.H., F31NS096863, E.A.B.).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, The normally hermaphrodite-specific PHB > AVA and PHA > AVG synaptic connections fail to prune in post-dauer adult males, and the normally male-specific PHB > AVG and AVG > DA9 connections are pruned in post-dauer adult hermaphrodites. Top, red cytoplasmic axon label; middle, GRASP (PHB > AVA, PHB > AVG, AVG > DA9) or iBLINC signal (PHA > AVG); bottom, magnified inset of colour-inverted synaptic puncta with arrowheads to indicate puncta. Intestinal auto-fluorescence is labelled ‘gut’. Representative images shown; for quantification and replication, see Fig. 1b, c and Methods. b, Starvation does not affect expression of the cell-specific promoters used for GRASP, and thus the effects on synaptic pruning are not an artefact of changes in promoter expression. Representative maximum intensity projection images of control animals and animals recovered from 24 h of L1 starvation are shown. mu, muscle (srg-13p is variably expressed in some muscle cells in addition to PHA). c, Neither L1 heat shock (30 min at 35 °C) nor L1 osmotic stress (24 h on plates with 200mM NaCl) affects male-specific pruning of the PHB > AVA connection. Each dot represents one animal (n = number of animals, shown in each column), blue bars show median, black boxes represent quartiles and vertical black lines show range. c, d, P values calculated by two-sided Wilcoxon rank-sum test with Bonferroni corrections for multiple testing (where applicable; see Methods). d, L1 starvation does not affect the normally male-specific PHB > AVG and AVG > DA9 synapses. Control animals are the progeny of starved animals.
a, SDS avoidance response is unchanged in day 2 adults. Left, predicted synaptic input into avoidance behaviour by relevant amphid and phasmid neurons1,5. Each dot represents the average reversal index of one animal over ten experimental trials, median shown with vertical magenta bar. P values calculated by two-sided Wilcoxon rank-sum test. b, Male mate-searching behaviour is unaffected by L1 starvation. Each dot represents the average distance one male travelled away from a bacterial lawn at four time points over 12 h (n = 48 animals control, 20 animals L1 starved), in the absence of hermaphrodites. Magenta bars indicate median, black boxes indicate quartiles. P values calculated by two-tailed t-test. By contrast, we did find mate-searching defects in adult males following recovery from dauer, suggesting that these males may have additional changes to the nervous system (data not shown).
Extended Data Fig. 3 Effects of starvation and exogenous or endogenous monoamine signalling on synaptic connectivity.
a, The normally hermaphrodite-specific PHB > AVA and PHA > AVG synaptic connections fail to prune in adult males following L1 starvation or treatment with exogenous octopamine during L1, but can be rescued by exogenous serotonin during L1 starvation. Top, red cytoplasmic axon label; middle, GRASP (PHB > AVA) or iBLINC signal (PHA > AVG); bottom, magnified inset of colour-inverted synaptic puncta with arrowheads to indicate puncta. Scale bars, 10 µm, all panels. Representative images shown; for quantification and replication, see Fig. 3a and Methods. b, Increases in extrasynaptic 5-HT through fluoxetine suppresses the failure to prune synapses. Quantification of PHB > AVA synaptic connectivity in adults following exposure to fluoxetine during L1 (on food), L1 starvation without fluoxetine, or L1 starvation in the presence of exogenous fluoxetine (0.1 mg ml–1). Each dot represents one animal (n = number of animals, shown in each column), blue bars show median, black boxes represent quartiles, vertical black lines show range (b, d). P values calculated by two-sided Wilcoxon rank-sum test with Bonferroni corrections for multiple testing (where applicable; see Methods). c, The effect of L1 starvation on male-specific synaptic pruning is rescued in the mod-5 mutant background. Representative images shown; for quantification and replication, see Fig. 3b and Methods. d, Loss of dopamine production (in a mutant for the cat-2 tyrosine hydroxylase) has no effect on the pruning of the PHB > AVA and PHA > AVG connections in males.
a, Time-course of tbh-1 transcriptional levels in fed (solid lines) and L1-starved (dashed lines) animals. tbh-1 levels are even higher after 24 h of starvation than after 12 h of starvation, providing a molecular correlate for our observation that 12 h of starvation is insufficient to affect male-specific synaptic pruning (Fig. 1d). Larval stages (and hours post-hatching for fed animals or post-transfer to food for starved animals at which imaging took place) shown on x-axis. Centre indicates median, error bars indicate quartiles (a, e). P values calculated by two-sided Wilcoxon rank-sum test (a–e). n = number of animals (shown below data points for fed, above for L1-starved) (a, e). b, Expression of a tph-1 transcriptional fosmid in NSM in fed L1 animals, starved L1 animals or L1 animals fed in the presence of 20 mg ml–1 exogenous octopamine. Each grey dot represents averaged expression level in one animal. Magenta bar indicates median, black box represents quartiles (b–d). n = number of animals (shown in each column) (b–d). c, Expression of a tph-1 transcriptional fosmid is not affected in ADF or NSM neurons (NSM data not shown) by exogenous tyramine in fed L1 hermaphrodites and males. TA, tyramine. d, tph-1 transcript levels quantified by smFISH. Maximum intensity projection images of one half of animal to show one NSM and one ADF neuron. Merge, overlay of tph-1 smFISH puncta onto DAPI. Number of tph-1 smFISH puncta was normalized to number of ric-4/SNAP-25 synaptic protein smFISH puncta in the same neuron to control for staining fluctuations, each dot (n = ) one neuron, shown in each column. e, Time-course of tph-1 transcriptional levels in fed (solid lines) and L1-starved (dashed lines) animals. Larval stages (and hours post-hatching for fed animals or post-transfer to food for starved animals at which imaging took place) shown on x-axis. Asterisks (fed L3 animals) indicate that animals were imaged at different laser settings (60% of all other time points) to prevent pixel oversaturation in images: thus, we under-estimate the magnitude of the L3 serotonin spike here.
Extended Data Fig. 5 Serotonin is downregulated by starvation and functions downstream, but not upstream, of tbh-1 transcription.
a, tbh-1 is not required for the initial downregulation of tph-1 transcription in ADF upon L1 starvation, but is required for the persistence of this downregulation into the L3 stage. Neither initial tph-1 downregulation nor a starvation memory were apparent in NSM in a tbh-1 null mutant. In well-fed conditions, there is no significant difference between control and tbh-1 mutant animals. Centre indicates median, error bars indicate quartiles. Solid lines indicate continuously fed animals, dashed lines indicate L1-starved animals. n = number of animals, shown in each column (a–c). P values calculated by two-sided Wilcoxon rank-sum test (a–c). b, The ser-6 octopamine receptor is required during sexual maturation to maintain tph-1 transcription levels in ADF but not NSM under well-fed conditions, and upon starvation tph-1 transcription levels in the ser-6 mutant do not further decrease, supporting the necessity of ser-6 for proper ADF starvation response. Magenta bar indicates median, black box represents quartiles (b, c). c, Upregulation of tbh-1 transcription upon L1 starvation is unaffected by the addition of exogenous serotonin (5 mM), suggesting that serotonin does not act upstream of tbh-1 upregulation (and subsequent octopamine production).
a, Effects of L1 starvation on male-specific synaptic pruning can also be rescued by exogenous serotonin during L3 (while animals are feeding). Each dot represents one adult male animal (n = number of animals, shown in each column), blue bar represents median, black box represents quartiles, vertical black bars represent range (a, b, e). P values calculated by two-sided Wilcoxon rank-sum test (a, b, e). b, tph-1 overexpression in NSM does not rescue starvation effects on pruning. Quantification of PHB > AVA and PHA > AVG synaptic connectivity in L1-starved adult males overexpressing tph-1 in NSM. Two independent transgenic lines were tested for each experiment; L1-starved animals without transgenic lines are siblings of transgenic animals; controls are non-starved adult males with transgenic arrays. None of the transgenic lines resulted in partial or complete rescue. c, Overexpression of tph-1 under an ADF-specific promoter during L1 starvation rescues the male-specific pruning of the PHB > AVA and PHA > AVG synaptic connections. Representative images shown; for quantification and replication, see Fig. 3e and Methods. d, tph-1 (n4622) and ttx-3 (ot22); unc-86 (n846) mutants (in which the NSM neuron does not express tph-1 or produce serotonin29) have cell body displacement, dendrite, and axon fasciculation defects in the phasmids. Overexpression of tph-1 under an NSM-specific promoter in the tph-1 (n4622) mutant background rescues the severity and penetrance of these defects. Representative images of defects in the PHB neuron are shown here as inverted black and white fluorescence images. Asterisk indicates dendrite defect, arrow shows anteriorly shifted cell body, arrowhead shows fasciculation defect. Scale bars, 10 µm. Per cent of animals with visible defects categorized and quantified to the right, n = number of animals, shown in each column. P values calculated by Freeman–Halton extension of one-sided Fisher exact test. e, Overexpression of tph-1 under an NSM-specific promoter in the tph-1 (n4622) mutant background (essentially, an ADF-specific tph-1 null) results in male-specific PHB > AVA pruning defects. Of two independent NSM::tph-1 transgenic lines, one resulted in a slight but insignificant defect in pruning, and one resulted in a substantial defect in pruning.
a, The normally hermaphrodite-specific PHB > AVA and PHA > AVG synaptic connections fail to prune in ser-4 mutant males, but pruning can be rescued by cell-specific expression of ser-4 cDNA in PHB or PHA. Top, red cytoplasmic axon label; middle, GRASP (PHB > AVA) or iBLINC signal (PHA > AVG); bottom, magnified inset of colour-inverted synaptic puncta with arrowheads to indicate puncta. Scale bars, 10 µm, all panels. Representative images shown; for quantification and replication, see Fig. 4c and Methods. b, ser-4 smFISH puncta are present in PHB in both sexes at L1. Three consecutive individual z-slices taken from the maximum intensity projections in Fig. 4b are shown, moving laterally through each animal from top to bottom rows. Dotted circles outline the PHB nuclei (DAPI) identified using osm-6::gfp (see Fig. 4b). Arrowheads in the top row indicate the locations of ser-4 puncta, which fade out of focus as the slices progress laterally. For quantification and replication, see Fig. 4b and Methods.
Extended Data Fig. 8 ser-4 is necessary for PHB > AVA and PHA > AVG synaptic pruning and acts downstream of the ADF serotonin signal.
a, Starvation does not enhance the male synaptic pruning defect in ser-4 mutants, and the ser-4 mutant phenotype cannot be rescued by exogenous serotonin. Each dot represents one animal (n = number of animals, shown in each column), blue bar represents median, black box represents quartiles, vertical black bars represent range. P values calculated by two-sided Wilcoxon rank-sum test (a, b). b, Expression of a tph-1 transcriptional fosmid in NSM and ADF does not significantly differ between wild-type and ser-4 mutant L1 animals. Each dot represents the expression level of one animal, n = number of animals, shown in each column. Magenta bar indicates median, black box represents quartiles.
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Bayer, E.A., Hobert, O. Past experience shapes sexually dimorphic neuronal wiring through monoaminergic signalling. Nature 561, 117–121 (2018). https://doi.org/10.1038/s41586-018-0452-0
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