Effects of sex and insulin/insulin-like growth factor-1 signaling on performance in an associative learning paradigm in Caenorhabditis elegans

Abstract

Learning is an adaptive change in behavior in response to environmental stimuli. In mammals, there is a distinct female bias to learn skills that is still unprecedented in other animal taxa. Here we have investigated the biological determinants of performance in an associative learning paradigm in the nematode Caenorhabditis elegans. Using an assay of chemotactic reactions associated with food deprivation, wild-type male worms show inferior learning ability relative to hermaphrodites. Sex-based learning difference is therefore an ancient evolutionary feature appearing even in relatively simple animals. C. elegans mutants with reduced insulin/IGF-1 signaling also exhibit a greatly reduced learning ability in this assay. In addition, hyperactivation of insulin/IGF-1 signaling through loss-of-function mutations in the PTEN phosphatase daf-18, a negative regulator of insulin/IGF-1 signaling, enhances learning ability beyond that of wild type. According to our epistasis analysis, the effect of DAF-2 on learning acts via phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) production, but not the DAF-16 FOXO transcription factor. This implies that the signaling pathway from DAF-2 affecting this learning paradigm branches between PIP(3) production and DAF-16. However, learning capacity of nematodes is lowered by loss-of-function mutations in daf-16, suggesting involvement of noninsulin/IGF-1 signaling-dependent DAF-16 activation in learning. Potentially, sex and insulin/IGF-1 signaling affect performance in this learning assay via effects on the neurobiology of learning.

Figure 1.—

Sex differences in the ability of young adult worms to modulate behavior. (A) Schematic of an assay plate. The shaded area indicates a local NaCl-containing region. Young adults were picked up individually from naive or conditioning plates, placed on the starting position of the assay plate, and left to move freely. The number of worms around each NaN₃ spot was counted. (B) Reduced capacity in starvation-induced suppression of chemotaxis of wild-type (N2) males as well as of him-8(e1489), him-5(e1490), and tra-1(e1099) mutant males. P < 0.001, except for him-5(e1490) males, where P < 0.01 (unpaired t-test). For the chemotaxis index (CI = N_N − N_C/N_T, where N_N is the number of animals around the NaCl gradient, N_C is the number of animals around the control spot, and N_T is the total number of animals), positive values demonstrate attraction to NaCl and negative values repulsion from NaCl. Worms were assayed for chemotaxis directly (naive) or conditioned by starving on a NGM plate either without (−) or with (+) NaCl for 4 hr. No mutants exhibited significant changes in behavioral response from the corresponding naives when they were conditioned on NaCl-free plates (data not shown). tra-1(e1575) gain-of-function mutants with XO karyotype are hermaphrodites whereas tra-1(e1099) loss-of-function mutant animals with XX karyotype are fertile males. (C) Chemotaxis of wild-type nematodes toward various concentrations of NaCl. At 0.1 mm NaCl, where only a fraction of nematodes responds to salt, males are still less repelled by salt than hermaphrodites (P < 0.01, unpaired t-test). Herm, hermaphrodite. Error bars indicate mean ± standard error.

Figure 1.—

Sex differences in the ability of young adult worms to modulate behavior. (A) Schematic of an assay plate. The shaded area indicates a local NaCl-containing region. Young adults were picked up individually from naive or conditioning plates, placed on the starting position of the assay plate, and left to move freely. The number of worms around each NaN₃ spot was counted. (B) Reduced capacity in starvation-induced suppression of chemotaxis of wild-type (N2) males as well as of him-8(e1489), him-5(e1490), and tra-1(e1099) mutant males. P < 0.001, except for him-5(e1490) males, where P < 0.01 (unpaired t-test). For the chemotaxis index (CI = N_N − N_C/N_T, where N_N is the number of animals around the NaCl gradient, N_C is the number of animals around the control spot, and N_T is the total number of animals), positive values demonstrate attraction to NaCl and negative values repulsion from NaCl. Worms were assayed for chemotaxis directly (naive) or conditioned by starving on a NGM plate either without (−) or with (+) NaCl for 4 hr. No mutants exhibited significant changes in behavioral response from the corresponding naives when they were conditioned on NaCl-free plates (data not shown). tra-1(e1575) gain-of-function mutants with XO karyotype are hermaphrodites whereas tra-1(e1099) loss-of-function mutant animals with XX karyotype are fertile males. (C) Chemotaxis of wild-type nematodes toward various concentrations of NaCl. At 0.1 mm NaCl, where only a fraction of nematodes responds to salt, males are still less repelled by salt than hermaphrodites (P < 0.01, unpaired t-test). Herm, hermaphrodite. Error bars indicate mean ± standard error.

Figure 1.—

Sex differences in the ability of young adult worms to modulate behavior. (A) Schematic of an assay plate. The shaded area indicates a local NaCl-containing region. Young adults were picked up individually from naive or conditioning plates, placed on the starting position of the assay plate, and left to move freely. The number of worms around each NaN₃ spot was counted. (B) Reduced capacity in starvation-induced suppression of chemotaxis of wild-type (N2) males as well as of him-8(e1489), him-5(e1490), and tra-1(e1099) mutant males. P < 0.001, except for him-5(e1490) males, where P < 0.01 (unpaired t-test). For the chemotaxis index (CI = N_N − N_C/N_T, where N_N is the number of animals around the NaCl gradient, N_C is the number of animals around the control spot, and N_T is the total number of animals), positive values demonstrate attraction to NaCl and negative values repulsion from NaCl. Worms were assayed for chemotaxis directly (naive) or conditioned by starving on a NGM plate either without (−) or with (+) NaCl for 4 hr. No mutants exhibited significant changes in behavioral response from the corresponding naives when they were conditioned on NaCl-free plates (data not shown). tra-1(e1575) gain-of-function mutants with XO karyotype are hermaphrodites whereas tra-1(e1099) loss-of-function mutant animals with XX karyotype are fertile males. (C) Chemotaxis of wild-type nematodes toward various concentrations of NaCl. At 0.1 mm NaCl, where only a fraction of nematodes responds to salt, males are still less repelled by salt than hermaphrodites (P < 0.01, unpaired t-test). Herm, hermaphrodite. Error bars indicate mean ± standard error.

Figure 2.—

Mutant worms with decreased insulin/IGF-1 signaling are defective in associative learning. (A) Regulatory hierarchy among the components of the insulin/IGF-1 genetic cascade in C. elegans. Arrows indicate activations, bars indicate negative regulatory interactions. DAF-2, the worm insulin/IGF-1 receptor; AGE-1, phosphatidylinositol-3-OH kinase (PI3K); DAF-18, PTEN phosphatase—the only known negative regulator of insulin/IGF-1 signaling; PDK-1, AKT-1, AKT-2, and SGK-1, phosphoinositide-dependent serine/threonine kinases; DAF-16, FOXO forkhead transcription factor. (B) Wild-type and insulin/IGF signaling-defective mutant hermaphrodites maintained at 20° were shifted to 25° and assayed for chemotaxis directly (naive) or conditioned on food-free NGM plates with NaCl. None of these strains displayed significant changes in behavior from the corresponding naives when they were conditioned on NaCl-free plates (data not shown). A genetic null mutation in daf-18, nr2037, improves the ability of hermaphrodites to change behavior. Error bars indicate mean ± standard error.

Figure 2.—

Mutant worms with decreased insulin/IGF-1 signaling are defective in associative learning. (A) Regulatory hierarchy among the components of the insulin/IGF-1 genetic cascade in C. elegans. Arrows indicate activations, bars indicate negative regulatory interactions. DAF-2, the worm insulin/IGF-1 receptor; AGE-1, phosphatidylinositol-3-OH kinase (PI3K); DAF-18, PTEN phosphatase—the only known negative regulator of insulin/IGF-1 signaling; PDK-1, AKT-1, AKT-2, and SGK-1, phosphoinositide-dependent serine/threonine kinases; DAF-16, FOXO forkhead transcription factor. (B) Wild-type and insulin/IGF signaling-defective mutant hermaphrodites maintained at 20° were shifted to 25° and assayed for chemotaxis directly (naive) or conditioned on food-free NGM plates with NaCl. None of these strains displayed significant changes in behavior from the corresponding naives when they were conditioned on NaCl-free plates (data not shown). A genetic null mutation in daf-18, nr2037, improves the ability of hermaphrodites to change behavior. Error bars indicate mean ± standard error.

Figure 3.—

The sex bias in associative learning in C. elegans is suppressed by mutations in daf-2. P < 0.001 (unpaired t-test). Error bars indicate mean ± standard error.

Figure 4.—

Mutations in the PTEN phosphatase daf-18 increase the ability of nematodes to modulate behavior. The daf-18 null mutation nr2037 restores the ability of males to change behavior to levels comparable with those of mutant hermaphrodites. P < 0.22 (unpaired t-test). nr2037 partially suppresses the learning-defective phenotype of daf-2(e1370) males. P < 0.01 (unpaired t-test). Herm, hermaphrodite. Error bars indicate mean ± standard error.