Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Mar 30, 2010; 107(13): 6016–6021.
Published online Mar 10, 2010. doi:  10.1073/pnas.1000866107
PMCID: PMC2851914
Neuroscience

Nuclear entry of a cGMP-dependent kinase converts transient into long-lasting olfactory adaptation

Abstract

To navigate a complex and changing environment, an animal's sensory neurons must continually adapt to persistent cues while remaining responsive to novel stimuli. Long-term exposure to an inherently attractive odor causes Caenorhabditis elegans to ignore that odor, a process termed odor adaptation. Odor adaptation is likely to begin within the sensory neuron, because it requires factors that act within these cells at the time of odor exposure. The process by which an olfactory sensory neuron makes a decisive shift over time from a receptive state to a lasting unresponsive one remains obscure. In C. elegans, adaptation to odors sensed by the AWC pair of olfactory neurons requires the cGMP-dependent protein kinase EGL-4. Using a fully functional, GFP-tagged EGL-4, we show here that prolonged odor exposure sends EGL-4 into the nucleus of the stimulated AWC neuron. This odor-induced nuclear translocation correlates temporally with the stable dampening of chemotaxis that is indicative of long-term adaptation. Long-term adaptation requires cGMP binding residues as well as an active EGL-4 kinase. We show here that EGL-4 nuclear accumulation is both necessary and sufficient to induce long-lasting odor adaptation. After it is in the AWC nucleus, EGL-4 decreases the animal's responsiveness to AWC-sensed odors by acting downstream of the primary sensory transduction. Thus, the EGL-4 protein kinase acts as a sensor that integrates odor signaling over time, and its nuclear translocation is an instructive switch that allows the animal to ignore persistent odors.

Keywords: neuron, plasticity, signaling, memory, integration

Sensory neurons, the entry point for information about an animal's external environment, must both respond to relevant stimuli and dampen their response as a consequence of prolonged stimulation. This dampening or adaptation is thought to be a protective consequence of prolonged stimulation, because indeed, adaptation protects mammalian photoreceptors from stimulation-induced degeneration (1, 2). The cellular, molecular, and temporal controls that allow sensory neurons to switch from being responsive to refractory to a given stimulus are examined herein. The Caenorhabditis elegans AWC sensory neuronal pair can sense (3) and adapt (4) to inherently attractive odors, and although both sensory and interneurons within the olfactory circuit adapt to persistent stimulation (5, 6), this focus will be on the events that instruct long-lasting adaptation within the sensory neuron.

The AWC neurons (3, 7) are postulated to express at least 20 different odor-responsive G protein-coupled receptors (8, 9), use cGMP as a second messenger (1013), and hyperpolarize in response to odor (14). Odor adaptation begins within 10 min of odor exposure, and there is a mild decrease in the animal's attraction to the odor accompanied by an increase in phosphorylated MAP kinase in AWC (5, 15) that requires the G protein gamma, GPC-1 (16). After 30 min of exposure, attraction decreases further as the animal enters a phase of short-term adaptation that requires the protein kinase (PKG) EGL-4 and a consensus PKG phosphorylation site within the C terminus of the cGMP-gated channel β-subunit, TAX-2 (17). After 60–90 min of odor exposure, the animal enters a long-term phase of adaptation in which it ignores the odor (4, 17) for at least 2.5 h (18). When starvation is coupled with 120 min of odor exposure, a new repulsive behavior is evoked (19). In this phase, the AWC neuron allows the animal to respond to butanone, but it switches from directing forward movement to initiating backward movement in response to the odor (19).

Genetic analysis has produced a list of factors required for adaptation (4, 6, 16, 17, 2024); however, the process by which these factors promote adaptation is unknown. To understand how adaptation proceeds in the AWC neuron, we complemented genetic methods with cell biological techniques. Specifically, we asked if localization of the EGL-4 kinase changes as a function of odor exposure and if this change might instruct long-term adaptation. Using a GFP tagged form of EGL-4, we found that prolonged odor exposure induces translocation of the PKG into the AWC nucleus and that this nuclear translocation is both necessary and instructive for long-term adaptation. After it is in the nucleus, EGL-4 is likely to alter the physiology of the neuron downstream of sensory transduction. Thus, we show that the PKG EGL-4 acts to sense and integrate odor signals over time to produce a lasting decrease in odor-seeking behavior.

Results

GFP-Tagged EGL-4 Enters the AWC Nucleus After Odor Exposure.

EGL-4 is required within the AWC neuron for long-term adaptation (Fig. 1A) (17). Odor-exposed animals lacking EGL-4 [egl-4(n479)] (Fig. S1A) exhibited the same chemotaxis index (CI) to benzaldehyde, an AWC sensed odor, as naive egl-4(n479) animals (P = 0.59) (Fig. 1A). In contrast, animals that were pre-exposed to benzaldehyde had a significantly lower CI than wild-type naive animals (P = 0.006) (Fig. 1A) (4, 17).

Fig. 1.
EGL-4 nuclear localization corresponds with behavior and is restricted to the odor-stimulated AWC. (A) Wild type (WT; N2, no transgene), egl-4(n479), egl-4(n479)+podr-3::GFP–EGL-4 (null animals expressing GFP–EGL-4 from the odr-3 promoter ...

We hypothesized that the subcellular localization of EGL-4 might both change as a function of prolonged odor exposure and yield a molecular tool with which to probe adaptation. To visualize endogenous EGL-4, antiserum against EGL-4 was used to stain fixed and permeabilized animals that had been exposed to buffer or benzaldehyde for 80 min. An odor-dependent accumulation of endogenous EGL-4 within the AWC nuclei was observed (Fig. S1). The specificity of our antiserum in whole-mount worms was confirmed as the staining pattern of egl-4(n479) null animals was indistinguishable from that of the preimmune serum's staining of wild-type animals (25). To visualize dynamic changes of EGL-4 localization in large populations, we fused a GFP moiety to the amino terminus of EGL-4 and placed this fusion under the control of the odr-3 promoter, which drives expression in the AWA, AWB, and AWC neurons and lower expression in ASH (26). This expression construct was integrated into chromosome V.

This construct was functional, because it rescued the adaptation defects of the egl-4(n479) null mutant strain (Fig. 1A). Furthermore, expression of the GFP–EGL-4 fusion in the wild-type genetic background did not significantly alter chemotaxis of buffer (P = 0.42) or odor-exposed animals (P = 0.75) compared with wild-type animals that did not express the fusion (Fig. 1A). Thus, GFP-tagged EGL-4 was not expressed at levels that alter chemosensory functions. Unless otherwise mentioned, all subsequent GFP–EGL-4 analysis was performed on animals that expressed GFP–EGL-4 from the integrated transgene and were wild type at the egl-4 locus.

EGL-4–GFP was evenly distributed throughout the cytoplasm of the AWC but was predominantly nuclear in the AWA and AWBs of naïve animals (Fig. 1 B and C). Eighty minutes of exposure to dilute benzaldehyde (0.01%) resulted in nuclear accumulation of GFP–EGL-4 within the AWCs (Fig. 1C). Thus, prolonged exposure to an AWC-sensed odor led to nuclear accumulation of GFP–EGL-4.

Nuclear Accumulation of GFP–EGL-4 Is Specific for the Odor-Stimulated AWC Neuron.

AWC nuclear accumulation was specific for AWC-sensed odors, because exposure to odors sensed by other neurons did not result in accumulation of GFP–EGL-4 within the AWC nucleus. The AWA neurons, another pair of sensory neurons, are used to track attractive volatiles such as diacetyl (3, 8). Prolonged exposure to diacetyl caused adaptation of the AWA-mediated chemotaxis response (Fig. 1E Left) (18) but did not affect the subcellular localization GFP–EGL-4 within AWC (Fig. 1E Right).

Benzaldehyde is sensed by both AWCs, whereas butanone is sensed only by the AWCON neuron that also expresses the str-2 promoter (27, 28). Butanone preexposure decreased attraction to butanone (Fig. 1F Left) and induced nuclear accumulation of GFP–EGL-4 in only one AWC neuron (Fig. 1F Right). By contrast, GFP–EGL-4 accumulated in both AWC neurons after benzaldehyde exposure (Fig. 1F Right). Butanone exposure led to nuclear accumulation of GFP–EGL-4 only within the nucleus of the butanone-responsive AWCON cell (marked with DsRed) (Fig. 1G). Thus, nuclear localization of EGL-4 was specific for the stimulated AWC.

Odor Exposure Decreased Odor-Seeking Behavior and Increased Nuclear Accumulation of GFP–EGL-4.

Having developed a visual reporter for EGL-4 localization, we were able to quantify nuclear accumulation and correlate it with behavioral changes over the time course of adaptation. We assessed both the CI (Fig. 2A Left) as well as the subcellular localization of GFP–EGL-4 (Fig. 2A Right) within the same population of animals (Fig. 1D for paradigm) as a function of the duration of odor exposure. We found that as the duration of odor exposure increased, there was an inverse correlation between nuclear accumulation of GFP–EGL-4 and CI to benzaldehyde (Fig. 2A). The CI decreased in a linear fashion, whereas the increase in percent of animals with nuclear EGL-4 was exponential. To quantify these differences, we examined the mean rate of decrease of odor-seeking behavior [the decrement of the CI within each trial (n = 5) per minute over each time interval, denoted as dCI/dt in Fig. 2A] and compared that to the rate of increase in the percent nuclear GFP–EGL-4 (denoted as d%nuc/dt in Fig. 2A). We found that the CI decreased in a linear fashion (such that the rate of decay or the change in CI per unit time between each time point did not significantly differ over the time course) (Fig. 2A Left Inset), whereas the increase in mean percent nuclear GFP–EGL-4 showed a significant jump (P = 0.04) between the 45- and 60-min time points compared with the initial 0- to 10-min rate (Fig. 2A Inset shows change in percent nuclear GFP–EGL-4 per unit time at each time interval). This inflection point in the rate of nuclear accumulation allowed us to identify a demarcation between early and late adaptation.

Fig. 2.
Nuclear EGL-4 correlates with a stable adaptation of AWC. (A) Animals were exposed to dilute benzaldehyde for the indicated amount of time before being assessed for CI to benzaldehyde (Left) or for GFP–EGL-4 localization within AWC (Right). Inset ...

Nuclear Localization of EGL-4 Coincides with Persistent Adaptation of the AWC-Mediated Olfactory Response.

We postulated that the quality of the adaptation might be different before and after the demarcation. One property of plasticity that changes with duration of stimulation in other systems is its stability. Thus, we decided to determine whether or not the stability of adaptation was altered in the early (0–30′ odor exposure) compared with the later (60–80′ odor exposure) phase of adaptation.

We asked whether or not the early phase of behavioral adaptation that is accompanied by a low percent of the population exhibiting nuclear GFP–EGL-4 would be more quickly reversed than the late phase that is accompanied by a large percent of the population exhibiting nuclear GFP–EGL-4. We exposed the strain expressing GFP–EGL-4 to benzaldehyde for either 30 or 80 min to induce either early or late adaptation, respectively. The early-adapted animals were then recovered in buffer alone for either 0 or 60 min, and the late-adapted ones for 0 or 150 min. After recovery, a portion of each population was subjected to chemotaxis assays, and another portion was subjected to microscopic analysis. The 30-min odor exposure led to a significant (P = 0.024) decrease in CI (Fig. 2B Upper, left pair of bars). The decrease was transient; by 60 min of recovery, the animals were as attracted to benzaldehyde as the unexposed controls (P = 0.135) (Fig. 2B Upper, right pair of bars). Thus, early adaptation that occurs while EGL-4 is cytoplasmic is transient and readily reversible.

Exposure to benzaldehyde for 80 min, however, decreased attraction to benzaldehyde 4-fold compared with buffer-treated animals (P = 0.002) (Fig. 2C Upper, left pair of bars), and this decrease persisted. After 150 min of recovery, the animals remained as unresponsive to benzaldehyde as those with no recovery period (Fig. 2C Upper). Importantly, the prolonged exposure to buffer alone (total of 230 min with exposure and recovery) did not affect the CI (P = 0.800) (Fig. 2C Upper). Immediately after the 80-min exposure, over 60% of the exposed population exhibited nuclear GFP–EGL-4 (Fig. 2C Lower, left light gray bar had 0′ recovery). This indicates that late adaptation accompanied by nuclear accumulation of GFP–EGL-4 can persist for at least 150 min.

Interestingly, GFP–EGL-4 left the nuclei of animals that had been allowed to recover for 150 min (P = 0.963) (Fig. 2C Lower, light gray bar had 150′ recovery). This was surprising, because these animals’ CI was as low as the CI of animals that had enjoyed no recovery period (P = 0.105) (Fig. 2C Upper). This indicates that after EGL-4 is concentrated within the AWC nucleus, the neuron becomes refractory to odor stimulation; additionally, this state persists even after EGL-4 has exited the nucleus. Thus, nuclear EGL-4 is required to initiate rather than to maintain odor adaptation. In sum, observation of the GFP-tagged reporter identified an inflection point in nuclear accumulation that predicted a difference in the quality of short- versus long-term adaptation, and it also allowed us to understand that the effect of nuclear EGL-4 persists after it exits the nucleus.

Both Short- and Long-Term Adaptation Required cGMP Binding and an Intact Kinase Domain.

To understand the domains of EGL-4 that are required for each phase of adaptation, residues within the GFP-tagged EGL-4 were altered (Fig. 3A) and expressed in egl-4(n479) animals from extrachromosomal arrays (equivalent levels of expression were assured by assessing GFP–EGL-4 intensity). We found that when we deleted the nuclear localization signal (NLS), GFP–EGL-4(ΔNLS) was exclusively cytoplasmic even after odor exposure (Figs. 3B and and44A), indicating that the NLS is required for nuclear entry and that the GFP–EGL-4(ΔNLS) fusion protein is not cotransported into the nucleus with another protein. As seen previously (17), expression of the NLS-deficient EGL-4 in egl-4(n479) animals allowed for some adaptation, although it was less than provided by wild-type EGL-4 (P = 0.05 comparing the wild-type benzaldehyde preexposed CI to that of the ΔNLS) (Fig. 3C).

Fig. 3.
Adaptation and nuclear accumulation of GFP–EGL-4 require cGMP binding, an NLS, and a kinase domain. (A) Domain structure of EGL-4 containing two cGMP-binding domains (merged), an NLS, and kinase domain. Mutations in the low-affinity (T276) and ...
Fig. 4.
Nuclear EGL-4 is necessary and sufficient for long-term olfactory adaptation of the AWCs. (A) egl-4(n479) animals expressing podr-3::GFP–EGL-4(ΔNLS) {from an extrachromosomal array indicated as egl-4(n479);Ex[podr-3::GFP-EGL-4(ΔNLS)]} ...

Two allosteric cGMP-binding domains are present in the amino one-half of EGL-4 (29, 30). In other PKGs, cGMP binding has been shown to relieve an inhibitory interaction between an amino terminal pseudosubstrate region and the kinase domain (31). cGMP binding has been shown to both activate the kinase and promote nuclear translocation of PKG 1α in mammalian cells (32). Altering key residues within either or both cGMP-binding domains (T276A and T398A) of GFP–EGL-4 led to exclusively cytosolic GFP, even after prolonged odor exposure (Fig. 3B). This suggests that EGL-4 requires cGMP binding for nuclear entry. These animals were completely defective for adaptation (Fig. 3C), indicating that both nuclear and cytoplasmic functions of EGL-4 depend on cGMP binding.

When we mutated a conserved aspartic acid that confers kinase activity to PKGs (D611N), odor-induced nuclear entry of GFP–EGL-4(D611N) was reduced (Fig. 3B). The NLS of the kinase-defective mutant should be exposed by cGMP binding (32), and yet, it is defective for nuclear entry. Since the NLS of the kinase defective mutant should be exposed by cGMP binding, the fact that we observe a partial dependence on the kinase domain indicates that nuclear translocation may be enhanced by a kinase function. Although some EGL-4(D611) entered the nucleus, it was unable to promote adaptation (Fig. 3C). Thus, EGL-4's kinase activity is required both in the cytoplasm and the nucleus.

Nuclear Accumulation of GFP–EGL-4 Is Necessary for Persistent Adaptation.

To probe whether or not nuclear accumulation is necessary for long-lasting adaptation, we asked if the adaptation seen in egl-4(n479); GFP–EGL-4(ΔNLS) was easily reversed or persistent. GFP–EGL-4(ΔNLS) was localized exclusively within the cytoplasm even after prolonged odor exposure (Figs. 3B and and4A).4A). GFP–EGL-4(ΔNLS) was functional, because it rescued the short-term adaptation defects when expressed in the egl-4(n479) strain (Fig. 4B Upper Left). Both wild-type [N2;Is(podr-3::GFP-EGL-4)] and EGL-4(ΔNLS)-expressing animals (egl-4(n479); Ex[podr-3::GFP-EGL-4(ΔNLS)]) were able to recover from short-term adaptation (Fig. 4B). Thus, EGL-4(ΔNLS), although unable to enter the nucleus, promotes easily reversed short-term adaptation.

We then asked whether or not long-term, persistent adaptation (Fig. 2C) actually requires nuclear EGL-4. Either wild-type EGL-4 or EGL-4(ΔNLS) was expressed in egl-4(n479) from an extrachromosomal array, and we found that each strain was able to adapt to 80 min of benzaldehyde exposure (Fig. 4B Lower Left). After 60 min of recovery, however, the wild-type EGL-4 expressing animals were still adapted, whereas the EGL-4(ΔNLS) expressing animals were not (Fig. 4B Lower Right). The recovery of the EGL-4(ΔNLS)-expressing animals was similar to that in animals that were exposed to odor for only 30 min (Figs. 2B and 4B Upper). Thus, long-lasting adaptation will not occur unless EGL-4 can enter the nucleus. This suggests that none of the other events evoked by odor exposure, such as G protein signaling or alterations in calcium levels, were sufficient to evoke long-term olfactory adaptation in animals that could not accumulate EGL-4 in the AWC nucleus and that nuclear entry of EGL-4 is necessary for long-lasting odor adaptation.

Nuclear Accumulation of GFP–EGL-4 Is Sufficient to Induce Long-Term Adaptation.

Next, we asked if nuclear localization of EGL-4 is sufficient to render animals unresponsive to AWC-sensed odors. We constructed a constitutively nuclear EGL-4 by adding a second NLS to the amino terminus of the GFP-tagged EGL-4 (NLS-GFP–EGL-4). This was expressed under the odr-3 promoter. Fluorescence expression was concentrated in the nuclei of all three pairs of neurons. AWC is shown in Fig. 4C.

EGL-4 is required in the AWA neuron for chemotaxis toward the odorant diacetyl (33). To determine whether or not the NLS-GFP–EGL-4 was functional, we asked if it could rescue the defective AWA-mediated chemotaxis of the egl-4(n479) strain. Expression of NLS-GFP–EGL-4 allowed the egl-4(n479) strain to respond to diacetyl in a manner that was close to the wild-type strain (Fig. 4D). This rescue was similar to that seen in Daniels et al. (33) with the un-tagged EGL-4. Thus, EGL-4 is functional even in the context of an additional NLS.

The response to the AWC-sensed odors, benzaldehyde, isoamyl alcohol, or butanone, however, was severely diminished in wild-type animals expressing NLS-GFP–EGL-4 (Fig. 4E). EGL-4's ability to diminish chemotaxis was dependent on the additional NLS, because the expression of an equivalent level of a wild-type GFP–EGL-4 did not alter the CI toward benzaldehyde (Fig. 4E and Fig. S2 for normalization). Thus, nuclear EGL-4 is sufficient to down-regulate AWC-mediated chemotaxis even in the absence of prior odor exposure. These results argue that nuclear accumulation of EGL-4 is an instructive event that stably dampens the animal's AWC olfactory response for hours.

Nuclear EGL-4 Acts Downstream of Primary Sensory Signal Transduction to Alter the AWC Neurons' Response to Odor.

In one model, nuclear EGL-4 could down-regulate all aspects of AWC function to silence the AWC neuron completely; in another model, the effect could be specific to the adapting odor. We asked if animals that had nuclear EGL-4 as a result of prolonged exposure to benzaldehyde could still respond to the second AWC-sensed odor isoamyl alcohol. Isoamyl alcohol is similar to benzaldehyde in that it caused nuclear accumulation of GFP–EGL-4 in both AWCs after prolonged exposure. Although GFP-EGL-4 was nuclear in both AWCs of a benzaldehyde-exposed starved population (Fig. 5A Upper), their response to isoamyl alcohol was the same as buffer-treated worms (Fig. 5A Lower). This amount of benzaldehyde was sufficient to cross-adapt isoamyl-alcohol responses in fed populations (4, 18) (Fig. S3). Thus, despite the fact that nuclear EGL-4 adapts the response to benzaldehyde, the isoamyl-alcohol response in the same neurons remains intact. This observation indicates that odor-induced EGL-4 nuclear entry does not completely eliminate primary sensory transduction or synaptic transmission in the AWC neurons.

Fig. 5.
Nuclear EGL-4 blocks chemotaxis downstream of primary odor sensation. (A Upper) Starved wild-type animals expressing podr-3::GFP–EGL-4 from an extrachromosomal array were exposed to buffer plus benzaldehyde (dark bars) or buffer (light bars) for ...

To determine the level at which EGL-4 affects AWC's response to the adapting stimulus itself, we turned to the paradigm of butanone exposure-induced butanone repulsion (19). Briefly, prolonged (120 min) butanone exposures cause starving animals to be repulsed from butanone. In this paradigm, butanone produces sensory stimulation of AWC, but synaptic communication with the downstream circuit is altered such that the butanone-sensing AWC drives repulsion instead of mediating attraction. We asked if EGL-4 is nuclear under circumstances where AWC mediates repulsion. If it is, it would indicate that nuclear EGL-4 does not block butanone odor sensation.

To understand whether or not GFP–EGL-4 was functional in this paradigm, we asked if EGL-4 was required in the AWC neuron for butanone repulsion. We found that it was; egl-4(n479) animals were not repulsed by butanone, but GFP-tagged EGL-4 rescued both the butanone sensation and repulsion to wild-type levels (Fig. 5B Lower). We saw that GFP–EGL-4 was indeed nuclear (Fig. 5B Upper) in the str-2ON AWC neurons of a large proportion of the animals that were repulsed from butanone (Fig. 5B Lower), but it was cytoplasmic in the same neuron of naive animals. This result indicates that nuclear EGL-4 does not block primary butanone sensation, because these animals are repulsed from butanone. Thus, nuclear EGL-4 acts downstream of primary sensory signal transduction within the AWC.

Next, we asked if nuclear EGL-4 is required for AWC to switch from attraction to repulsion in this paradigm. Indeed, starved, butanone-exposed egl-4(n479) animals expressing EGL-4(ΔNLS) were not repulsed by butanone (Fig. 5B Lower). Importantly, these animals were attracted to butanone before exposure and showed short-term levels of adaptation, indicating that EGL-4(ΔNLS) was functional enough to restore the cytoplasmic but not nuclear functions of EGL-4. Thus, the switch between attraction to and repulsion from butanone requires nuclear EGL-4. Taken together, our results indicate that nuclear EGL-4 does not block primary odor sensation but may act at another level to either dampen or alter the quality of the AWC sensory signal in both long-term adapted and butanone-repulsed animals.

Nuclear EGL-4 Alters Gene Expression.

Phosphorylation of EGL-4 nuclear targets could alter transcription, splicing, nuclear import, export, or translation (e.g. via drosha). We first asked whether or not nuclear EGL-4 altered transcription. In contrast to mammalian PKGs, we found no evidence that either the cAMP response element-binding protein (CREB) (24, 34) or transcription from the fos promoter was a target of nuclear EGL-4 (35) (Fig. S4). However, transcription of the STR-2 member of the G-protein coupled receptor (GPCR) class of genes was up-regulated by nuclear EGL-4 (Table S1). Prior work had shown that expression of STR-2 is restricted to either the right or left AWC neuron (27), requires guanylyl cyclases ODR-1 and DAF-11 as well as EGL-4 (36), and is redundantly activated by G-alpha proteins ODR-3 and either GPA-2 or GPA-6 (37). When the promoter of STR-2 (27) driving DsRed was employed as a visual reporter for pstr-2 expression, we found that expression was lost in an egl-4(ks60) loss-of-function mutant (Table S1). The ks60 allele removes critical C-terminal residues required for EGL-4’s function as a kinase. This indicates that STR-2 expression requires EGL-4. Nuclear EGL-4 was required for str-2 expression, because the constitutively cytoplasmic EGL-4(ΔNLS) was unable to induce reporter expression in egl-4(n479) mutants (Table S1). Furthermore, we found that induction of NLS-GFP–EGL-4 from the heat-shock promoter (animals were heated for 2 h before analysis) was sufficient to promote reporter expression in adult egl-4(n479, null) animals. However, we failed to observe adaptation-dependent changes in the transcription of a dynamic str-2 reporter (Fig. S5). This could indicate that either our reporter was not sensitive enough to detect odor-dependent changes in expression or str-2 transcription is not altered in the course of adaptation. These data indicate that nuclear entry of EGL-4 can induce GPCR expression in the adult AWC neuron. Thus, in long-term adaptation, nuclear accumulation of EGL-4 is likely to direct changes in transcription that stably alter the physiology of the AWC

Discussion

Examination of EGL-4’s localization in the process of adaptation has revealed that nuclear accumulation of EGL-4 switches adaptation from being labile to persistent. In short-term adaptation, EGL-4 acts within the cytoplasm where the activity of its targets may be maintained by phosphorylation, thereby allowing dephosphorylation to rapidly return their activity to that of the naïve state. By contrast, the long-lasting effects of nuclear EGL-4 could result from phosphorylation of transcription factors or chromatin that stably change expression of downstream factors. Where these downstream factors act and how they might affect the physiology of the neuron is unknown. We do, however, have evidence that nuclear EGL-4 does not abrogate AWC's ability to sense and respond to all odors, because long-term benzaldehyde adaptation does not affect isoamyl-alcohol chemosensation. Furthermore, although EGL-4 is nuclear in starved and butanone-exposed animals, these animals are repulsed by butanone. Thus, enough sensory signaling must be present to allow the animals to avoid this odor even when EGL-4 is nuclear. Thus, nuclear EGL-4 may alter expression of genes that ultimately affect synaptic function. In a separate study, we provide genetic evidence that several potentially synaptic factors act downstream of nuclear EGL-4 to promote adaptation (24).

Experimental Procedures

Strains and Maintenance.

Bristol N2 was used as our wild-type strain, and egl-4(n479), egl-4(ks60), and pyIs500[(p)odr-3::GFP::EGL-4] (see below) were used in this study. Strains were maintained according to standard protocols (38).

Plasmid Construction and Transgenic Strains.

Standard molecular and C. elegans techniques were used to generate plasmids and strains (SI Experimental Procedures).

Odor-Related Behavioral Assays.

Chemotaxis was performed as described in ref. 3, and adaptation was performed as described in refs. 1719 (SI Experimental Procedures).

Microscopic Assessment of GFP–EGL-4 Subcellular Localization.

This was carried out on mounted anesthetized animals (SI Experimental Procedures).

Supplementary Material

Supporting Information:

Acknowledgments

The authors thank Kyoung-hye Yoon, Mehrdad Matloubian, Maria Gallegos, Amanda Khan-Kirby, Shih-Yu Chen, Mark Lucanic, Marie Burns, and Cori Bargmann for helpful comments on incarnations of this work, Mark Alkema, Renate Pilz, Michael Silberbach, and Darren Browning for helpful discussions, and Robert Bailey and Renee Engle for technical assistance. This work was supported by the National Institute on Deafness and Other Communication Disorders National Research Service Award F31DC007031 (to J.I.L.), National Institutes of Health Grant 5T32DC008072 (to J.A.K), National Science Foundation Graduate Fellowship Grant 2008073299 (to S.O.H.), Medical Scientist Training Program Grant GM07739 (to B.L.), National Institute of Mental Health National Research Service Award F30MH084482 (to B.L.), the University of California at San Francisco Program for Breakthrough Biomedical Research (A.G.), National Institutes of Health/National Institute on Deafness and Other Communication Disorders Grant R01 005991 (to N.D.L.), and the University of California at Davis’ Tupin and Health Sciences Award.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/1000866107/DCSupplemental.

References

1. Burns ME, Arshavsky VY. Beyond counting photons: Trials and trends in vertebrate visual transduction. Neuron. 2005;48:387–401. [PubMed]
2. Fain GL. Why photoreceptors die (and why they don't) Bioessays. 2006;28:344–354. [PubMed]
3. Bargmann CI, Hartwieg E, Horvitz HR. Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell. 1993;74:515–527. [PubMed]
4. Colbert HA, Bargmann CI. Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans. Neuron. 1995;14:803–812. [PubMed]
5. Hirotsu T, Iino Y. Neural circuit-dependent odor adaptation in C. elegans is regulated by the Ras-MAPK pathway. Genes Cells. 2005;10:517–530. [PubMed]
6. Matsuki M, Kunitomo H, Iino Y. Goalpha regulates olfactory adaptation by antagonizing Gqalpha-DAG signaling in Caenorhabditis elegans. Proc Natl Acad Sci USA. 2006;103:1112–1117. [PMC free article] [PubMed]
7. White JG, Southgate E, Thomson JN, Brenner S. The nervous system of the nematode, Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci. 1986;314:1–340. [PubMed]
8. Sengupta P, Chou JH, Bargmann CI. odr-10 encodes a seven transmembrane domain olfactory receptor required for responses to the odorant diacetyl. Cell. 1996;84:899–909. [PubMed]
9. Troemel ER, Chou JH, Dwyer ND, Colbert HA, Bargmann CI. Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell. 1995;83:207–218. [PubMed]
10. Birnby DA, et al. A transmembrane guanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate a common set of chemosensory behaviors in caenorhabditis elegans. Genetics. 2000;155:85–104. [PMC free article] [PubMed]
11. Coburn CM, Bargmann CI. A putative cyclic nucleotide-gated channel is required for sensory development and function in C. elegans. Neuron. 1996;17:695–706. [PubMed]
12. Komatsu H, Mori I, Rhee JS, Akaike N, Ohshima Y. Mutations in a cyclic nucleotide-gated channel lead to abnormal thermosensation and chemosensation in C. elegans. Neuron. 1996;17:707–718. [PubMed]
13. L'Etoile ND, Bargmann CI. Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase ODR-1. Neuron. 2000;25:575–586. [PubMed]
14. Chalasani SH, et al. Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans. Nature. 2007;450:63–70. [PubMed]
15. Hirotsu T, Saeki S, Yamamoto M, Iino Y. The Ras-MAPK pathway is important for olfaction in Caenorhabditis elegans. Nature. 2000;404:289–293. [PubMed]
16. Yamada K, Hirotsu T, Matsuki M, Kunitomo H, Iino Y. GPC-1, a G protein gamma-subunit, regulates olfactory adaptation in Caenorhabditis elegans. Genetics. 2009;181:1347–1357. [PMC free article] [PubMed]
17. L’Etoile ND, et al. The cyclic GMP-dependent protein kinase EGL-4 regulates olfactory adaptation in C. elegans. Neuron. 2002;36:1079–1089. [PubMed]
18. Colbert HA, Bargmann CI. Environmental signals modulate olfactory acuity, discrimination, and memory in Caenorhabditis elegans. Learn Mem. 1997;4:179–191. [PubMed]
19. Tsunozaki M, Chalasani SH, Bargmann CI. A behavioral switch: cGMP and PKC signaling in olfactory neurons reverses odor preference in C. elegans. Neuron. 2008;59:959–971. [PMC free article] [PubMed]
20. Palmitessa A, et al. Caenorhabditus elegans arrestin regulates neural G protein signaling and olfactory adaptation and recovery. J Biol Chem. 2005;280:24649–24662. [PubMed]
21. Miyahara K, Suzuki N, Ishihara T, Tsuchiya E, Katsura I. TBX2/TBX3 transcriptional factor homologue controls olfactory adaptation in Caenorhabditis elegans. J Neurobiol. 2004;58:392–402. [PubMed]
22. Ikeda DD, et al. CASY-1, an ortholog of calsyntenins/alcadeins, is essential for learning in Caenorhabditis elegans. Proc Natl Acad Sci USA. 2008;105:5260–5265. [PMC free article] [PubMed]
23. Kaye JA, Rose NC, Goldsworthy B, Goga A, L'Etoile ND. A 3’UTR pumilio-binding element directs translational activation in olfactory sensory neurons. Neuron. 2009;61:57–70. [PubMed]
24. O’Halloran DM, Altshuler-Kyelin S, Lee JI, L’Etoile ND. Regulators of AWC-mediated olfactory plasticity in Caeorhabditis elegans. PLoS Genet. 2009;5:1000761. Available at: http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1000761, Accessed December 5, 2009. [PMC free article] [PubMed]
25. Eastham-Anderson JR. Davis, CA: University of California; 2004. Nuclear translocation of EGL-4, a cGMP dependant (i.e. dependent) protein kinase, in response to odorant adaptation in C. elegans. Master’s thesis.
26. Roayaie K, Crump JG, Sagasti A, Bargmann CI. The G alpha protein ODR-3 mediates olfactory and nociceptive function and controls cilium morphogenesis in C. elegans olfactory neurons. Neuron. 1998;20:55–67. [PubMed]
27. Troemel ER, Sagasti A, Bargmann CI. Lateral signaling mediated by axon contact and calcium entry regulates asymmetric odorant receptor expression in C.elegans. Cell. 1999;99:387–398. [PubMed]
28. Wes PD, Bargmann CI. C. elegans odour discrimination requires asymmetric diversity in olfactory neurons. Nature. 2001;410:698–701. [PubMed]
29. Reed RB, et al. Fast and slow cyclic nucleotide-dissociation sites in cAMP-dependent protein kinase are transposed in type Ibeta cGMP-dependent protein kinase. J Biol Chem. 1996;271:17570–17575. [PubMed]
30. Stansberry J, et al. A cGMP-dependent protein kinase is implicated in wild-type motility in C. elegans. J Neurochem. 2001;76:1177–1187. [PubMed]
31. Francis SH, Corbin JD. Structure and function of cyclic nucleotide-dependent protein kinases. Annu Rev Physiol. 1994;56:237–272. [PubMed]
32. Gudi T, Lohmann SM, Pilz RB. Regulation of gene expression by cyclic GMP-dependent protein kinase requires nuclear translocation of the kinase: Identification of a nuclear localization signal. Mol Cell Biol. 1997;17:5244–5254. [PMC free article] [PubMed]
33. Daniels SA, Ailion M, Thomas JH, Sengupta P. egl-4 acts through a transforming growth factor-beta/SMAD pathway in Caenorhabditis elegans to regulate multiple neuronal circuits in response to sensory cues. Genetics. 2000;156:123–141. [PMC free article] [PubMed]
34. Lu YF, Kandel ER, Hawkins RD. Nitric oxide signaling contributes to late-phase LTP and CREB phosphorylation in the hippocampus. J Neurosci. 1999;19:10250–10261. [PubMed]
35. Gudi T, Casteel DE, Vinson C, Boss GR, Pilz RB. NO activation of fos promoter elements requires nuclear translocation of G-kinase I and CREB phosphorylation but is independent of MAP kinase activation. Oncogene. 2000;19:6324–6333. [PubMed]
36. Lesch BJ, Gehrke AR, Bulyk ML, Bargmann CI. Transcriptional regulation and stabilization of left-right neuronal identity in C. elegans. Genes Dev. 2009;23:345–358. [PMC free article] [PubMed]
37. Lans H, Rademakers S, Jansen G. A network of stimulatory and inhibitory Galpha-subunits regulates olfaction in Caenorhabditis elegans. Genetics. 2004;167:1677–1687. [PMC free article] [PubMed]
38. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...