Regulation of Diacylglycerol Content in Olfactory Neurons Determines Forgetting or Retrieval of Olfactory Memory in Caenorhabditis elegans

dgk-1 mutation suppressed the forgetting defect of the tir-1 loss-of-function mutant

In C. elegans, WT animals show attractive chemotaxis to various odorants. It has been reported that low diacetyl concentrations are mainly sensed by AWA olfactory neurons, whereas high diacetyl concentrations are also sensed by AWC neurons (Bargmann et al., 1993; Sengupta et al., 1996; Chou et al., 2001). While naive animals were strongly attracted to diacetyl (Fig. 1A, Naive), after preexposure to 0.02% diacetyl for 90 min without food, animals showed olfactory adaptation in which they changed their behavior to show weaker chemotaxis to diacetyl (Bargmann et al., 1993; Colbert and Bargmann, 1995) (Fig. 1A, Adaptation). We studied this behavioral plasticity as a model of memory acquisition of diacetyl and starvation (Inoue et al., 2013; Hadziselimovic et al., 2014; Kitazono et al., 2017). After adaptation, WT animals restored the chemotaxis to diacetyl within 4 h to the level of naive animals, which is considered as “forgetting” (Inoue et al., 2013; Hadziselimovic et al., 2014) (Fig. 1A, Recovery in WT). We have previously found that a tir-1 loss-of-function (lf) mutant (tm3036) shows prolonged retention of the memory (Fig. 1A) and the TIR-1/JNK-1 pathway, which functions in the AWC olfactory neurons, accelerates forgetting of the memory of diacetyl in AWA neurons (Inoue et al., 2013). However, the molecular and neuronal mechanisms by which the TIR-1/JNK-1 pathway in AWC neurons regulates forgetting the memory in the AWA neurons is still unknown.

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Figure 1.

dgk-1 mutation suppressed the forgetting defect of the tir-1 loss-of-function mutant. A, Suppression of the forgetting defect of the tir-1 mutant by dgk-1 mutation. Chemotaxis of naive, adapted, and 4 h recovered animals was analyzed. Asterisks indicate significant differences between after adaptation and after 4 h recovery. WT: p = 3.35 × 10⁻⁷; tir-1;dgk-1(qj203): p = 4.29 × 10⁻⁸ (n = 6, F_(4,45) = 9.98, p = 7.18 × 10⁻⁶). B, Transgenic rescue of tir-1(tm3036);dgk-1(qj203) animals expressing WT dgk-1 genomic fragments. Asterisks indicate significant differences between after adaptation and after 4 h recovery or between recovery with or without dgk-1 fragment. WT: p = 2.84 × 10⁻¹⁷; tir-1;dgk-1(qj203) without dgk-1 fragment: p = 5.23 × 10⁻¹⁰; recovery with or without dgk-1 fragment: p = 4.08 × 10⁻⁵ (n = 10, F_(6,108) = 22.22, p = 6.72 × 10⁻¹⁷). C, dgk-1(nu62) and D, dgk-1(sy428) mutations also suppressed tir-1's forgetting defect. Asterisks indicate significant differences between after adaptation and after 4 h recovery. C, WT: p = 2.32 × 10⁻¹⁵; tir-1;dgk-1(nu62): p = 9.38 × 10⁻⁶ (n ≥ 10, F_(4,135) = 17.42, p = 1.50 × 10⁻¹¹). D, WT: p = 1.53 × 10⁻¹⁵; tir-1;dgk-1(sy428): p = 0.000247 (n = 8, F_(4,63) = 22.51, p = 1.40 × 10⁻¹¹). E, Representative images of the GFP-tagged ODR-10 protein at cilia of AWA in WT and mutants. Scale bar, 10 μm. F, Quantitative analyses of fluorescent intensities of expression of the GFP-tagged ODR-10 protein at cilia. Statistics: one-way ANOVA with Tukey–Kramer multiple test. Asterisks indicate significant differences between dgk-1(qj203) and others. dgk-1(qj203) versus WT: p = 9.14 × 10⁻¹³; versus tir-1(tm3036): p = 9.37 × 10⁻¹³; versus tir-1;dgk-1: p = 9.52 × 10⁻¹³ (n = 72, 77, 64, 134, F_(3,343) = 44.42, p = 2.82 × 10⁻²⁴). G, Phenotype of the dgk-1(qj203) single mutant. Chemotaxis of naive, adapted, and 4 h recovered animals was analyzed. Asterisks indicate significant differences between after adaptation and after 4 h recovery. WT: p = 1.44 × 10⁻⁹; dgk-1(qj203): p = 5.09 × 10⁻⁹ (n = 8, F_(4,63) = 6.67, p = 0.000153). H, dgk-1 also suppresses the forgetting defect of the tir-1 mutant, even, when attracted by an even lower concentration (1:1000) of diacetyl. Asterisks indicate significant difference between naive and after recovery. tir-1(tm3036): p = 8.91 × 10⁻⁵ (n = 12, F_(6,132) = 4.06, p = 0.000907). I, dgk-1 only modestly, but significantly, suppresses the forgetting defect in tir-1 in the case of the memory of isoamyl alcohol. Asterisks indicate significant differences between after adaptation and after recovery or naive and after recovery. Adaptation versus recovery in WT: p = 2.54 × 10⁻¹⁸; tir-1(tm3036): p = 9.96 × 10⁻¹²; dgk-1(qj203): p = 7.76 × 10⁻²⁶; tir-1;dgk-1: p = 3.03 × 10⁻⁵. Naive versus recovery in tir-1: p = 1.17 × 10⁻²⁷; tir-1;dgk-1: p = 2.11 × 10⁻⁹ (n = 12, F_(6,132) = 45.34, p = 8.98 × 10⁻³⁰). J, tir-1 does not show a forgetting defect in the memory of pyrazine. Asterisks indicate significant differences between after adaptation and after recovery. WT: p = 3.73 × 10⁻¹⁴; tir-1(tm3036): p = 3.36 × 10⁻¹⁰; dgk-1(qj203): p = 3.36 × 10⁻⁹; tir-1;dgk-1: p = 3.35 × 10⁻⁷ (n = 6, F_(6,60) = 2.04, p = 0.0744). Box plots: center line indicates median. Box range, 25th-75th percentiles. Whiskers represent minimum-maximum values. Dots represent the data points. External dots from whisker represent outliers. **p < 0.01; ***p < 0.001; two-way ANOVA with Bonferroni correction (except F).

In this study, to explore new components downstream of the TIR-1/JNK-1 pathway, we first performed an unbiased suppressor screening of the tir-1 (tm3036 null) forgetting defect phenotype. In this screening, we identified several suppressor mutations that restored the forgetting phenotype. Using whole genome sequencing and single nucleotide polymorphism analyses, we found that, among the identified mutants, qj203 mutation in dgk-1, which encodes DAG kinase-1, suppressed the defective forgetting phenotype in the tir-1 mutant (Fig. 1A). This suppression could be rescued by the WT dgk-1 gene, indicating this dgk-1 mutation (qj203) is responsible for the suppression (Fig. 1B). This mutation causes a premature stop codon (W646) to stop, which is the same as the n892 allele in the DAG kinase catalytic domain (Nurrish et al., 1999; Jose and Koelle, 2005). We also showed that the other dgk-1 mutants, dgk-1(nu62) and dgk-1(sy428), both of which are regarded as null mutants (Hajdu-Cronin et al., 1999; Nurrish et al., 1999; Jose and Koelle, 2005), suppressed the forgetting defect of the tir-1 mutant, like dgk-1(qj203) (Fig. 1C,D). Although the double mutants tir-1;dgk-1 showed slightly lower chemotaxis to diacetyl (Fig. 1A–D), we could not detect significant differences in expression of a GFP-tagged ODR-10 protein, a receptor for diacetyl, between WT and tir-1;dgk-1(qj203) (Fig. 1E,F), suggesting that the expression of ODR-10 receptor does not affect the naive chemotaxis. Although the single mutant dgk-1(qj203) showed significantly higher intensity than other strains (Fig. 1F), the single mutation did not affect chemotaxis in naive, adaptation, and forgetting (Fig. 1G). These results suggest that the function of dgk-1 itself is not required for chemotaxis, adaptation to diacetyl, and its forgetting, but it is required for the regulation of forgetting downstream of tir-1.

Confirming that the effect of the dgk-1 mutations is involved in the forgetting process in AWA neurons, we found that dgk-1 suppressed the forgetting defect of tir-1, even when attracted to lower concentrations (10⁻³) of diacetyl of which sensing is thought to be more restricted to AWA neurons (Sengupta et al., 1996; Chou et al., 2001) (Fig. 1H). Next, to examine whether forgetting of the memory for odorants other than diacetyl was regulated by the same pathway, we analyzed the behavioral forgetting of the memory for isoamyl alcohol, which is predominantly sensed by AWC neurons, and pyrazine, which is predominantly sensed by AWA neurons (Bargmann et al., 1993). We found that the forgetting defect of the tir-1 mutant in the memory of isoamyl alcohol was only modestly suppressed by the dgk-1 mutation (Fig. 1I), while, for pyrazine, even in tir-1 mutant, the forgetting defect was not observed (Fig. 1J), suggesting that the forgetting of the olfactory adaptation is differently regulated depending on the type of odorants.

DGK-1, a human DGKθ homolog, depletes DAG by changing it into phosphatidic acid (PA) via phosphorylation (Houssa et al., 1997; Nurrish et al., 1999; Van Blitterswijk and Houssa, 2000). DAG is an important second messenger in various biological processes, including facilitation of synaptic release (Hajdu-Cronin et al., 1999; Lackner et al., 1999; Nurrish et al., 1999; Miller et al., 2000; Van Blitterswijk and Houssa, 2000) (Fig. 2A). In C. elegans, DAG controls various types of behavior, such as locomotion (Nurrish et al., 1999; Miller et al., 2000), egg-laying (Hajdu-Cronin et al., 1999), thermotaxis (Biron et al., 2006; Nakano et al., 2020), odor chemotaxis (Tsunozaki et al., 2008), and learning of salt and food (Adachi et al., 2010; Kunitomo et al., 2013; Ohno et al., 2017).

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Figure 2.

DGK-3, EGL-30 (Gαq), and GOA-1 (Gαo) are also involved in the regulation of forgetting downstream of the TIR-1/JNK-1 pathway. A, A model of the DAG signaling pathway based on Nurrish et al. (1999); Lackner et al. (1999); and Miller et al. (2000). DAG is produced by EGL-8 (PLCβ) downstream of EGL-30, which is inhibited by GOA-1, activates protein kinase C and UNC-13, and results in enhancing synaptic transmission. DGK-1 degrades DAG to PA. B, Suppression of the forgetting defect of the tir-1 mutant by the DAG kinase dgk-3. Chemotaxis of naive, adapted, and 4 h recovered animals was analyzed. Asterisks indicate significant differences between after adaptation and after 4 h recovery. WT: p = 8.87 × 10⁻⁹; dgk-3(gk110): p = 5.76 × 10⁻¹⁴; tir-1; dgk-3: p = 8.10 × 10⁻⁶ (n = 6, F_(6,60) = 7.90, p = 2.73 × 10⁻⁶). C, Suppression of the forgetting defect of the tir-1 mutant by egl-30 gain-of-function mutation (ep271). WT: p = 2.51 × 10⁻¹⁵; egl-30(ep271 gf): p = 7.33 × 10⁻¹²; egl-30(gf);tir-1: p = 2.96 × 10⁻⁸ (n = 8, F_(6,84) = 22.48, p = 1.25 × 10⁻¹⁵). D, Suppression of the forgetting defect of the tir-1 mutant by goa-1 loss-of-function mutation (n1134). WT: p = 5.77 × 10⁻⁸; goa-1: p = 1.87 × 10⁻⁸; goa-1;tir-1: p = 0.000104 (n = 12, F_(6,132) = 8.50, p = 8.22 × 10⁻⁸). Box plots: center line indicates median. Box range, 25th-75th percentiles. Whiskers represent minimum-maximum values. Dots represent the data points. External dots from whisker represent outliers. ***p < 0.001 (two-way ANOVA with Bonferroni correction).

To examine whether the regulation of DAG content is important for the regulation of forgetting, we analyzed the tir-1-suppressive activity of the DAG kinase dgk-3, which controls thermotaxis as a thermal memory molecule (Biron et al., 2006) and adaptation to AWC-sensed odorants (Matsuki et al., 2006). We found that a dgk-3 (lf) mutation suppressed the forgetting defect of the tir-1 mutant (Fig. 2B), suggesting that multiple DAG kinases coordinately regulate forgetting by controlling the DAG content downstream of the TIR-1/JNK-1 pathway.

Two types of G-proteins are involved in the regulation of forgetting

In C. elegans, the G-proteins, EGL-30 (Gqα), and GOA-1 (Goα), function antagonistically in the regulation of the DAG content (Mendel et al., 1995; Segalat et al., 1995; Brundage et al., 1996; Hajdu-Cronin et al., 1999; Lackner et al., 1999) (Fig. 2A): EGL-30 upregulates DAG content, whereas GOA-1 downregulates it in motor neurons (Lackner et al., 1999; Nurrish et al., 1999; Miller et al., 2000) (Fig. 2A). Furthermore, EGL-30 and GOA-1 in AWC neurons regulate the adaptation to AWC-sensed odorants, but not to AWA-sensed odorants (Matsuki et al., 2006). Additionally, activation of Gqα signaling in AWC neurons enhances memory consolidation of the associative learning of butanone, an AWC-sensed odorant, with food (Arey et al., 2018). Therefore, we examined whether these G-proteins regulate forgetting of the olfactory memory by analyzing the behavioral phenotypes of an egl-30 gain-of-function (gf) mutant and a goa-1 (lf) mutant, which are considered to have higher DAG content, like the dgk-1 (lf) mutant.

The single mutants egl-30(ep271 gf) and goa-1(n1134 lf) did not exhibit significant differences in naive chemotaxis, adaptation, and forgetting, like the WT and dgk-1 single mutant (Fig. 2C,D). The double mutants with tir-1 revealed that both egl-30(gf) and goa-1 suppressed the forgetting defect of the tir-1 mutant, like tir-1;dgk-1 (Fig. 2C,D), suggesting that these G-proteins also regulate the forgetting process, probably through the DAG content. Our data suggest that this forgetting regulation by DAG signaling is also necessary for proper forgetting in WT animals (see below).

DAG regulates forgetting of the adaptation in the olfactory circuit

To determine where the DAG content-modulating molecules regulate forgetting, we performed cell-specific overexpression and rescue experiments. First, we tested whether the expression of egl-30(gf), a constitutively active form of egl-30, under a pan-neuronal, AWC, or AIA promoter suppresses the forgetting defect of the tir-1 mutant (Fig. 3A–C). Pan-neuronal expression of egl-30(gf) in the tir-1 mutant restored the chemotaxis after a 4 h recovery to the same extent as that of the naive nematode (Fig. 3A), although these animals showed lower chemotaxis in naive conditions. The expression of odr-1 promoter::egl-30(gf), which gives an expression predominantly in AWC sensory neurons (L'Etoile and Bargmann, 2000), suppressed the forgetting defect of the tir-1 mutant, without affecting its naive chemotaxis (Fig. 3B). On the other hand, the expression of gcy-28.d promoter::egl-30(gf), which gives an expression specifically in AIA interneurons that are downstream of AWA and AWC neurons (Tsunozaki et al., 2008; Chalasani et al., 2010), did not show significant suppression of the forgetting defect (Fig. 3C). These results suggest that activating EGL-30, which presumably resulted in the upregulation of the DAG content, accelerates forgetting, even in the absence of tir-1 function; thus, for proper forgetting, the activity of EGL-30 is downregulated in AWC neurons downstream of the TIR-1/JNK-1 pathway. Additionally, in naive condition, relatively weak chemotaxis in the double mutants of tir-1 with mutations, which elevate DAG (Figs. 1A, 2C,D), did not occur in the overexpression of egl-30 in AWC neurons (Fig. 3B), suggesting that their weak chemotaxis in naive is a side effect unrelated to the suppression of tir-1's forgetting defect.

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Figure 3.

The DAG content needs to be reduced in AWC and AIA neurons during forgetting. A–C, Transgenic rescue of tir-1(tm3036) mutants by expressing a constitutively active form of the egl-30 gene under a pan-neuronal (H20) (A), AWC (odr-1) (B), or AIA (gcy-28.d) promoter (C). Chemotaxis of naive, adapted, and 4 h recovered animals was analyzed. Asterisks indicate significant differences between after adaptation and after 4 h recovery. A, WT: p = 0.000465; tir-1;Ex[H20p::egl-30(gf)]: p = 0.00166 (n = 4, F_(6,36) = 7.81, p = 2.00 × 10⁻⁵). B, WT: p = 0.00136; tir-1;Ex[odr-1p::egl-30(gf)]: p = 4.40 × 10⁻⁵ (n = 6, F_(6,60) = 3.81, p = 0.00280). C, WT: p = 3.15 × 10⁻⁸ (n = 4, F_(6,36) = 6.40, p = 0.000118). D–G, Transgenic rescue of the goa-1(n1134);tir-1(tm3036) mutant's phenotype by expressing the WT goa-1 gene under its own (goa-1) (D), pan-neuronal (H20) (E), AWC (odr-1) (F), or AIA (gcy-28.d) promoter (G). D, WT: p = 8.07 × 10⁻³¹; goa-1;tir-1 (Ex-): p = 4.06 × 10⁻⁹ (n = 18, F_(6,204) = 22.73, p = 1.87 × 10⁻²⁰). E, WT: p = 2.79 × 10⁻⁶; goa-1;tir-1 (Ex-): p = 0.000961 (n = 8, F_(6,84) = 8.60, p = 2.71 × 10⁻⁷). F, WT: p = 1.09 × 10⁻⁷; tir-1 (tm3036): p = 0.00618; goa-1;tir-1 (Ex-): p = 0.000388 (n = 12, F_(6,132) = 11.30, p = 3.67 × 10⁻¹⁰). G, WT: p = 3.51 × 10⁻¹⁸; goa-1;tir-1 (Ex-): p = 8.36 × 10⁻⁶ (n = 8, F_(6,84) = 17.22, p = 6.99 × 10⁻¹³). H, Overexpression of goa-1 in AWC neurons prevents WT animals from forgetting. WT: p = 1.91 × 10⁻¹³; WT (Ex-): p = 2.62 × 10⁻⁶ (n = 8, F_(6,84) = 11.00, p = 5.16 × 10⁻⁹). Box plots: center line indicates median. Box range, 25th-75th percentiles. Whiskers represent minimum-maximum values. Dots represent the data points. External dots from whisker represent outliers. **p < 0.01; ***p < 0.001; two-way ANOVA with Bonferroni correction.

Second, we analyzed where goa-1 functions in the forgetting process (Fig. 3D–G). We found that, in goa-1;tir-1 double mutant in which the adaptation is recovered within a few hours, the expression of goa-1 cDNA under its own promoter or a pan-neuronal promoter caused the forgetting defect (Fig. 3D,E). The expression of goa-1 in AWC neurons or AIA neurons also caused the forgetting defect after a 4 h recovery (Fig. 3F,G). These data suggest that GOA-1, which presumably downregulates the DAG content, works not only in AWC but also AIA neurons to prevent forgetting.

As mentioned above, mutations that were expected to upregulate the DAG content may induce forgetting despite the tir-1 defect (Figs. 2, 3A–G). Consistently, downregulation of the DAG content was inferred to prevent forgetting. As expected, animals expressing goa-1(gf) (Q205L) in AWC neurons in a WT background, in which the DAG content in AWC neurons may decrease, still showed weak chemotaxis to diacetyl after the 4 h recovery, suggesting that they have a forgetting defect like the tir-1 mutant (Fig. 3H). These results indicate that the regulation of forgetting by changing the DAG content is not restricted to the absence of tir-1.

Finally, we examined where DGK-1 functions. The suppression of the forgetting defect in the tir-1 mutant by the lack of dgk-1 was rescued by dgk-1a cDNA expressed under a pan-neuronal promoter or ins-1(long) promoter, which expresses in multiple neurons (Kodama et al., 2006), including AWC and AIA (Hammarlund et al., 2018) (Fig. 4A,B). However, dgk-1.a expression in AWC neurons can slightly, but significantly, rescue the behavioral phenotype of tir-1;dgk-1 double mutant (Fig. 4C), although dgk-1.a expression in AIA neurons did not show the significant effect of the rescue (Fig. 4D). In contrast, the combined expression of both promoters was effective to rescue the suppression of tir-1's forgetting defect by dgk-1 (Fig. 4E). These findings suggest that DGK-1 functions simultaneously in AWC and AIA neurons in the regulation of forgetting. Together, changes in the DAG content in AWC sensory neurons and AIA interneurons probably regulate forgetting.

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Figure 4.

DGK-1 works in several neurons, including AWC and AIA in the regulation of forgetting. A–E, Transgenic rescue of the tir-1(tm3036);dgk-1(qj203) mutant's phenotype by expressing the WT dgk-1 gene isoform under its pan-neuronal (H20) (A), multiple neuronal (ins-1(long)) (B), AWC (odr-1) (C), AIA (gcy-28.d) (D) promoter, or AWC (odr-1) and AIA (gcy-28.d) promoter combined (E). Chemotaxis of naive, adapted, and 4 h recovered animals was analyzed. Asterisks indicate significant differences between after adaptation and after 4 h recovery or between naive and after 4 h recovery. A, WT: p = 2.60 × 10⁻²³; tir-1(tm3036): p = 0.00248; tir-1;dgk-1 (Ex-): p = 1.03 × 10⁻⁷ (n = 10, F_(6,108) = 17.52, p = 4.47 × 10⁻¹⁴). B, WT: p = 7.15 × 10⁻¹³; tir-1(tm3036): p = 0.00568; tir-1;dgk-1 (Ex-): p = 9.15 × 10⁻⁶ (n = 12, F_(6,132) = 6.73, p = 3.10 × 10⁻⁶). C, Adaptation versus recovery in WT: p = 1.58 × 10⁻²³; tir-1;dgk-1 (Ex-): p = 6.65 × 10⁻⁹; tir-1;dgk-1;Ex[odr-1p::dgk-1a]: p = 0.00232; and Naive versus recovery in tir-1(tm3036): p = 3.71 × 10⁻¹⁶; tir-1;dgk-1;Ex[odr-1p::dgk-1a]: p = 0.00191 (n = 10, F_(6,108) = 17.88, p = 2.63 × 10⁻¹⁴). D, Adaptation versus recovery in WT: p = 1.97 × 10⁻²¹; tir-1;dgk-1 (Ex-): p = 2.51 × 10⁻⁵; tir-1;dgk-1;Ex[gcy-28.dp::dgk-1a]: p = 0.000298; and Naive versus recovery in tir-1(tm3036): p = 5.37 × 10⁻²¹; tir-1;dgk-1;Ex[odr-1p::dgk-1a]: p = 0.000316 (n = 20, F_(6,228) = 15.11, p = 1.51 × 10⁻¹⁴). E, WT: p = 2.30 × 10⁻¹⁹; tir-1;dgk-1 (Ex-): p = 1.47 × 10⁻⁶ (n = 8, F_(6,84) = 18.72, p = 1.05 × 10⁻¹³). F, Effect of inhibiting synaptic transmission from AWC sensory neurons by expressing TeTx in goa-1;tir-1 mutants. Asterisks indicate significant differences between after adaptation and after 4 h recovery. WT: p = 1.84 × 10⁻⁵; goa-1;tir-1 (Ex-): p = 0.000260 (n = 12, F_(6,132) = 6.00, p = 1.42 × 10⁻⁵). G, Analyzing forgetting defect of olfactory adaptation in AWB-ablated (str-1p::mCasp-1), AWC-ablated (ceh-36p::caspase-3(p12)::nz + ceh-36p::cz::caspase-3(p17)), and AWB/C-ablated (odr-1p::mCasp1) animals. WT: p = 2.07 × 10⁻⁹; AWB (–): p = 2.48 × 10⁻¹⁸ (n = 6, F_(8,75) = 13.62, p = 5.51 × 10⁻¹²). Box plots: center line indicates median. Box range, 25th-75th percentiles. Whiskers represent minimum-maximum values. Dots represent the data points. External dots from whisker represent outliers. **p < 0.01; ***p < 0.001; two-way ANOVA with Bonferroni correction.

Regulation of the DAG content controls behavioral forgetting via synaptic transmission from AWC

In C. elegans, the DAG content is important for synaptic transmission (Nurrish et al., 1999; Miller et al., 2000) (Fig. 2A). We examined whether synaptic transmission is involved in the DAG-regulated forgetting process, using animals expressing Tetanus Toxin light chain (TeTx) to inhibit synaptic transmission (Schiavo et al., 1992). Inhibition of synaptic transmission from AWC neurons overrode the suppression of the forgetting defect of the tir-1 mutant by the goa-1 mutation in the goa-1;tir-1 double mutant (Fig. 4F).

Additionally, we examined whether AWB neurons are also required for proper forgetting because the odr-1 promoter used as an AWC promoter in this study has also been reported to be expressed in AWB sensory neurons (Yu et al., 1997). AWB-ablated animals did not show the forgetting defect, whereas AWC-ablated animals showed the forgetting defect (Fig. 4G). This result is consistent with our previous report on the ceh-36 mutant (Inoue et al., 2013); thus, synaptic transmission from AWC neurons, but not AWB neurons, are necessary for the suppression of tir-1 effect by dgk-1 and its upstream factor goa-1, and GOA-1 disturbs synaptic transmission from AWC neurons by modulating the DAG content to regulate forgetting (Fig. 2A). Accordingly, these results suggest that the rescues by expression by odr-1 promoters (Figs. 3B,F, 4C,E) are caused by expression in AWC neurons.

Behavioral forgetting can occur even without elimination of the memory traces in AWA sensory neurons

Our previous report revealed that the Ca²⁺ responses in AWA neurons change in parallel with the behavioral change in WT and tir-1 animals (Inoue et al., 2013). Because AWA is the primary sensory neuron for diacetyl, the diminished Ca²⁺ response after adaptation can be considered as a sensory memory trace for this plasticity. To examine whether the G-proteins and DAG are important for the maintenance or elimination of the memory trace, we analyzed the Ca²⁺ responses to diacetyl in AWA neurons of these strains. First, we confirmed the previously reported finding (Inoue et al., 2013; Kitazono et al., 2017) that in WT animals, Ca²⁺ responses to diacetyl in AWA neurons are evident under naive conditions, become weak after adaptation, and are recovered after a 4 h cultivation after adaptation (Fig. 5A,H). On the other hand, the Ca²⁺ responses in the tir-1 mutant were very similar to those in WT in the naive state and after adaptation, although after a 4 h recovery, the Ca²⁺ responses were still very weak (Fig. 5B,I), indicating that we reproduced the previous results and that the Ca²⁺ response can be considered as a sensory memory trace (Inoue et al., 2013). Additionally, ceh-36 mutants, which lack functional AWC neurons (Lanjuin et al., 2003), also had retained the memory trace after 4 h recovery in accord with the behavior similarly to tir-1 (Fig. 5C,J; Inoue et al., 2013; Fig. 4G, “AWC (–)”). Therefore, the forgetting defect in loss of tir-1 or AWCs is probably caused by inability to degrade the memory trace.

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Figure 5.

The Ca²⁺ response in AWA olfactory neurons is inconsistent with the behavior after a 4 h recovery of the goa-1;tir-1 and tir-1;dgk-1 double mutants, and the goa-1(gf)-expressing mutant. A–G, Quantification of the Ca²⁺ response of naive, adapted, and 4 h recovered animals. Averaged traces of the Ca²⁺ response in WT animals (n ≥ 18) (A), tir-1(tm3036) mutants (n ≥ 10) (B), ceh-36(ks86) mutants (n ≥ 12) (C), goa-1(n1134) mutants (n ≥ 10) (D), goa-1(n1134);tir-1(tm3036) double mutants (n ≥ 15) (E), tir-1(tm3036);dgk-1(qj203) double mutants (n ≥ 14) (F), and WT animals with overexpression of goa-1(gf) in AWC neurons (n ≥ 10) (G). Pink-colored background represents the timing of the diacetyl stimulation (from 20 to 50 s). R represents the YFP/CFP ratio, and R₀ represents the averaged ratio of 5 s before diacetyl stimulation. H–N, Peak values of the quantitated Ca²⁺ responses (A–G) in WT (H), tir-1(tm3036) (I), ceh-36(ks86) (J), goa-1(n1134) (K), goa-1(n1134);tir-1(tm3036) (L), tir-1(tm3036);dgk-1(qj203) (M) animals, and WT animals with overexpression of goa-1(gf) in AWC neurons (N). Asterisks indicate significant differences between after adaptation and after 4 h recovery. H, WT: p = 0.00182 (n = 18, 19, 23, F_(2,57) = 17.23, p = 1.40 × 10⁻⁶); I, tir-1: (n = 11, 10, 12, F_(2,30) = 9.75, p = 0.000548); J, ceh-36: (n = 13, 12, 14, F_(2,36) = 20.05, p = 1.41 × 10⁻⁶); K, goa-1: p = 0.0285 (n = 10, 14, 16, F_(2,37) = 16.97, p = 5.89 × 10⁻⁶); L, goa-1;tir-1: (n = 15, 15, 17, F_(2,44) = 9.92, p = 0.000278); M, tir-1;dgk-1: (n = 14, 15, 18, F_(2,44) = 32.68, p = 2.00 × 10⁻⁹); N, odr-1p::goa-1(gf): p = 0.0120 (n = 12, 10, 18, F_(2,37) = 5.40, p = 0.00877). Box plots: center line indicates median. Box range, 25th-75th percentiles. Whiskers represent minimum-maximum values. Dots represent the data points. External dots from whisker represent outliers. *p < 0.05; **p < 0.01; one-way ANOVA with Tukey–Kramer multiple test.

Next, we analyzed the change in the sensory response in the goa-1 single mutant, which showed similar behavioral phenotypes to WT in the naive and adaptation states, and after a 4 h recovery from the adaptation. We found that the Ca²⁺ responses in the goa-1 mutant were also very similar to those in the WT, which is consistent with its behavior (Fig. 5D,K) and in agreement with our hypothesis. Subsequently, we examined whether the Ca²⁺ responses in the goa-1;tir-1 double mutant are similar to those in the goa-1 mutant. We found that, in the goa-1;tir-1 double mutant, even after a 4 h recovery, the Ca²⁺ responses to diacetyl in the AWA neurons did not recover, which is similar to the response in the tir-1 mutant (Fig. 5E,L). These findings suggest that, in the goa-1;tir-1 double mutant, the sensory memory trace of adaptation is maintained even after a 4 h recovery. Furthermore, the Ca²⁺ responses in tir-1;dgk-1 double mutant also did not recover after 4 h cultivation (Fig. 5F,M) differently from the behavioral recovery (Fig. 1A). Together, in the goa-1; tir-1 and tir-1;dgk-1 mutants, although the sensory responses had been diminished even after a 4 h recovery from adaptation (Fig. 5E,F,L,M), the chemotaxis behaviors were recovered, and these animals behaved as if their memory traces were lost (Figs. 1A, 2D), suggesting that the DAG content can control the forgetting process in behavior without affecting the memory trace in the sensory response.

Overexpression of goa-1 in AWC neurons sustains the behavioral memory, even in the absence of a memory trace in AWA

We also analyzed the Ca²⁺ responses in AWA neurons in animals expressing goa-1(gf) in AWC neurons, which showed a behavioral forgetting defect (Fig. 3H). We found that the Ca²⁺ response to diacetyl recovered from adaptation after a 4 h cultivation similar to the WT animals (Fig. 5G,N). These results suggest that expressing goa-1(gf) in AWC neurons caused loss of the sensory memory trace in AWA neurons after a 4 h recovery; thus, the AWA neurons showed the Ca²⁺ responses to diacetyl like naive animals. Therefore, after a 4 h recovery from adaptation, despite the strong sensory response to diacetyl in the AWA neurons, the animals expressing goa-1(gf) in the AWC neurons exhibited weak chemotaxis as if they retained the memory traces. Together, the change in the DAG content in AWC neurons controls the forgetting process in behavior, but not the forgetting process in the sensory memory trace.

High DAG content until acquisition of memory and low DAG content after the acquisition lead to promote forgetting

To examine whether, as suggested by the genetic and imaging analyses, an increase of DAG really induces forgetting in tir-1 mutant, we administered PMA, an analog of DAG, to WT and tir-1 worms, during cultivation, conditioning, and/or recovery. In WT animals, the addition of PMA did not cause significant behavioral changes in all conditions (Fig. 6A). On the other hand, tir-1 mutant incubated with PMA during cultivation or during adaptation caused to show the attractive chemotaxis after 4 h recovery (Fig. 6B), suggesting that PMA until acquisition of memory leads to induce forgetting. However, tir-1 mutant exposed PMA during 4 h recovery, even if they exposed PMA during adaptation, showed forgetting defect (Fig. 6B). These results suggest that high DAG content before or during conditioning results in forgetting, even in tir-1 loss-of-function mutant, and that the higher DAG content after the conditioning cancelled this effect. Additionally, by cultivation with PMA, the naive chemotaxis to diacetyl of tir-1 mutant became weaker than that by cultivation without PMA (Fig. 6B). This result is consistent with the low chemotaxis of double mutants of tir-1 with mutation elevating DAG like dgk-1 (Figs. 1, 2). Moreover, the cultivation with PMA before conditioning, but not during adaptation, made the adaptation to diacetyl weaker in both of WT and tir-1 lines (Fig. 6A,B), suggesting that, similar to tir-1(gf) animals (Inoue et al., 2013), high DAG content before memory acquisition prevents acquisition.

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Figure 6.

High DAG content until memory acquisition and low DAG content after the acquisition leads to promote forgetting. A, B, The chemotaxis of naive, adapted, and 4 h recovery of PMA administered WT animals (A) and tir-1 mutant (B) was analyzed. –, exposed only solvent (ethanol as control); PMA, exposed PMA dissolved in ethanol. For example, PMA→PMA→ −, exposed to PMA dissolved in ethanol during cultivation and adaptation, and to ethanol during 4 h recovery. A, The chemotaxis of WT animals after recovery was not affected by PMA exposure during cultivation, during adaptation, and/or during recovery, individually. In addition, PMA exposure during cultivation made WT difficult to adapt to diacetyl. Asterisks indicate significant differences between after adaptation and after 4 h recovery or between after adaptation of two conditions. Adaptation versus recovery in − → − → −: p = 9.94 × 10⁻²⁰; PMA → − → −: p = 7.40 × 10⁻⁵; − → PMA → −: p = 5.44 × 10⁻¹⁸; − → − → PMA: p = 1.56 × 10⁻¹⁷; − → PMA → PMA: p = 3.52 × 10⁻¹²; and adaptation in − → − → − versus PMA → − → −: p = 1.43 × 10⁻⁸; PMA → − → − versus − → PMA → −: p = 7.13 × 10⁻⁷ (n = 8, F_(8,105) = 6.13, p = 1.81 × 10⁻⁶). B, tir-1 mutant exposed PMA during cultivation or during adaptation rose the chemotaxis of after recovery. However, PMA exposure during recovery canceled the effect. PMA exposure during cultivation reduced the naive chemotaxis of tir-1 and made it difficult to adapt to diacetyl. Asterisks indicate significant differences between naive, after adaptation, or after 4 h recovery of two conditions. Recovery in − → − → − versus PMA → − → −: p = 0.00281; − → − → − versus − → PMA → −: p = 2.77 × 10⁻⁵; − → PMA → − versus − → PMA → PMA: p = 2.18 × 10⁻⁶; naive in − → − → − versus PMA → − → −: p = 0.000633; adaptation in − → − → − versus PMA → − → −: p = 1.30 × 10⁻⁵; PMA → − → − versus − → PMA → −: p = 0.000217 (n = 8, F_(8,105) = 13.10, p = 5.91 × 10⁻¹³). C, Representative expression of DownwardDAG2 (Green) with DiI (Red) staining pattern in WT. DownwardDAG2 expressed in AWC neurons. Scale bar, 10 μm. D, Fluorescent intensity of DownwardDAG2 was measured in AWC sensory neurons of WT and mutants at naive, after adaptation, and after recovery. Asterisks indicate significant differences between strains in naive, after adaptation, or after 4 h recovery. In naive, WT versus tir-1: p = 0.00711 (n = 66, 63, 80, 65, F_(3,270) = 4.03, p = 0.00787). In adaptation, WT versus tir-1: p = 0.00946; WT versus goa-1;tir-1: p = 0.00360; tir-1 versus goa-1: p = 0.0315; goa-1 versus goa-1;tir-1: p = 0.0129 (n = 49, 44, 68, 53, F_(3,210) = 6.55, p = 0.000297). In recovery, WT versus tir-1: p = 2.68 × 10⁻⁵; tir-1 versus goa-1: p = 4.38 × 10⁻⁵ (n = 64, 56, 74, 45, F_(3,235) = 9.82, p = 3.97 × 10⁻⁶). Box plots: center line indicates median. Box range, 25th-75th percentiles. Whiskers represent minimum-maximum values. Dots represent the data points. External dots from whisker represent outliers. *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA with Bonferroni correction (A,B) or one-way ANOVA with Tukey–Kramer multiple test (D).

Furthermore, to examine whether DAG content in AWC neurons was distinct among strains and conditions, we monitored the DAG levels of AWC neurons at naive, after adaptation, and after recovery, in WT and the mutants by using a DAG indicator DownwardDAG2 (Tewson et al., 2012, 2013; Ohno et al., 2017) (Fig. 6C), of which lower DAG content increases fluorescent intensity. The fluorescent intensity of DownwardDAG2 in AWC neurons in tir-1 at naive exhibited higher than WT (Fig. 6D, left), suggesting that the DAG content in AWC neurons of tir-1 mutant is usually lower than that of WT. After adaptation, the fluorescent intensity of DownwardDAG2 in tir-1 remained higher than that in WT, and that in goa-1;tir-1 also became higher than in WT (Fig. 6D, middle). After 4 h recovery, tir-1 kept higher fluorescent intensity than WT (Fig. 6D, right). These results suggest that, although the DAG content in AWC neurons of tir-1 is constantly lower than that of WT, goa-1 loss of function brings about higher DAG content to tir-1 at least before memory acquisition (in naive) and leads to behavioral forgetting. Together, although the results of DownwardDAG2 expressed in AWC did not completely match with that of PMA administered assay (Fig. 6A,B), probably because administration of PMA affects DAG content of the whole body, a decrease in DAG content during until and after memory acquisition presumably gives rise to the behavioral forgetting.