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Nicotine, the primary addictive substance in tobacco, induces profound behavioral responses in mammals, but the underlying genetic mechanisms are not well understood. Here we develop a C. elegans model of nicotine-dependent behavior. We show that worms exhibit behavioral responses to nicotine that parallel those observed in mammals, including acute response, tolerance, withdrawal and sensitization. These nicotine responses require nicotinic acetylcholine receptor (nAChR) family genes that are known to mediate nicotine dependence in mammals, suggesting functional conservation of nAChRs in nicotine responses. Importantly, we find that mutant worms lacking TRPC (transient-receptor-potential canonical) channels are defective in response to nicotine and that such a defect can be rescued by a human TRPC channel, revealing an unexpected role for TRPC channels in regulating nicotine-dependent behavior. Thus, C. elegans can be used to characterize known genes as well as to identify new genes regulating nicotine responses.
Nicotine dependence is a worldwide health problem and represents the leading preventable cause of death in industrialized countries (Champtiaux and Changeux, 2004; Laviolette and van der Kooy, 2004). The primary molecular target of nicotine is nicotinic acetylcholine receptors (nAChRs), a family of pentameric calcium-permeable cation channels (Champtiaux and Changeux, 2004). In mammals, nicotine binds to nAChRs in the ventral tegmental area, leading to stimulation of the mesolimbic dopamine system (Laviolette and van der Kooy, 2004). While much is known about the role of nicotine in directly modulating the function of nAChRs, the genetic mechanisms by which such modulation leads to prolonged behavioral and neurological changes are poorly understood.
Due to their simple nervous system and amenability to genetic manipulation, invertebrate organisms such as C. elegans and Drosophila have been widely utilized as genetic models to study various phenomena in neurobiology, including substance dependence. For example, recent studies in Drosophila have provided new insights into the mechanisms of alcohol tolerance/intoxication and cocaine sensitivity, and work in C. elegans has identified novel players involved in alcohol intoxication (Andretic et al., 1999; Bainton et al., 2005; Davies et al., 2003; Scholz et al., 2005). The C. elegans genome encodes 28 nAChR genes, many of which have been shown to form functional nAChRs in heterologous systems (Jones and Sattelle, 2004). While the role of nicotine in regulating the activity of muscle cells (e.g. egg-laying and body-wall muscles) has been well characterized (Gottschalk et al., 2005; Lewis et al., 1980; Waggoner et al., 2000), the effects of nicotine on the function of the nervous system, which mediates nicotine dependence in vertebrates, have not been evaluated.
TRP (transient-receptor-potential) channels represent a superfamily of cation channels conserved from worms to humans and comprise seven subfamilies (TRPC, TRPV, TRPM, TRPN, TRPA, TRPP and TRPML) (Montell, 2005; Ramsey et al., 2006). The founding members of the TRP superfamily are the TRPC (TRP-Canonical) channels, which can be activated following the stimulation of phospholipase C and/or depletion of internal calcium stores (Montell, 2005). However, the precise mechanisms leading to TRPC activation remain unclear and somewhat controversial. Studies in cell culture systems have implicated TRPCs in a wide variety of physiological processes in mammals ranging from muscle relaxation/contraction, fluid secretion, growth cone guidance and morphology, to acrosome reaction (Montell, 2005; Ramsey et al., 2006). Nevertheless, the genetic evidence supporting such functions is rather limited.
In the current study, we developed a C. elegans model of nicotine-dependent behavior. We show that worms display acute and chronic behavioral responses to nicotine that parallel those observed in mammals. These responses require nAChRs that are known to be critical for nicotine dependence in mammals. We have also identified TRPC channels as novel proteins important for nicotine-dependent behavior. TRPC channels functionally regulate nicotine-induced cellular responses in the locomotion circuitry. Our results suggest that C. elegans can be used as a genetic model for identifying and characterizing neuronal genes regulating nicotine responses.
In rodent models, nicotine stimulates locomotor activity in naïve animals, though it initially induces transient hypoactivity (Dwoskin et al., 1999). Chronic nicotine treatment adapts animals to nicotine (Dwoskin et al., 1999; Laviolette and van der Kooy, 2004). In addition, nicotine cessation evokes withdrawal symptoms (Kenny and Markou, 2001). To quantify the effects of nicotine on worm locomotion, we used a worm tracking system that records worm locomotion and reports its activity, such as locomotion velocity, in real time (Li et al., 2006).
We first examined the locomotion behavior of naïve animals. After being transferred to a new environment (i.e. a fresh plate with bacteria), the animals exhibited a continuous decay in locomotion speed until reaching a relatively steady state (Figure 1A and S2D), consistent with previous observations (Zhao et al., 2003). However, when assayed on plates containing nicotine, the animals displayed a distinct locomotion behavior: After a brief rapid decay in locomotion speed, the animals then began to gradually speed up their locomotion (Figure 1B). We named this phenomenon “locomotion-stimulation”. The locomotion-stimulation phase, which was not seen in naïve animals, became evident starting at ~4 minutes after the acute nicotine incubation and perdured until the speed plateaued (Figure 1B). Such a response to nicotine was dose-dependent and peaked at around 1.5 μM (Figure 1E). Interestingly, the nicotine concentration in human blood peaks at ~500 nM after consumption of one cigarette (Pidoplichko et al., 1997).
We next tested the long-term effects of nicotine on locomotion and found that after chronic nicotine treatment, worms developed tolerance to nicotine in a time-dependent manner (Figure 1C, 1F and S2E). As a result, these nicotine-adapted animals behaved similarly to naïve animals on nicotine-free plates, demonstrating that chronic nicotine treatment elicits tolerance (adaptation) in C. elegans (Figure 1C and 1F).
To test the effects of nicotine withdrawal, we moved the animals that were chronically treated (>16 hrs) with nicotine to nicotine-free plates. In response to such nicotine cessation, these nicotine-adapted animals began to increase their locomotion speed at ~4 minutes after nicotine withdrawal and displayed a locomotion-stimulation phase similar to that observed in naïve animals responding to acute nicotine exposure (Figure 1D). This suggests that these nicotine-adapted animals became dependent on nicotine for their naïve-like behavior on nicotine plates (Figure 1D). These results indicate that nicotine cessation can induce withdrawal responses in C. elegans.
In vertebrates, repeated intermittent administrations of nicotine can sensitize an animal’s response to nicotine, a process believed to be critical for the development of nicotine dependence (Dwoskin et al., 1999; Laviolette and van der Kooy, 2004). To test whether worms can be sensitized to nicotine, we treated naïve animals for 20 minutes with a lower concentration of nicotine that did not induce significant locomotion-stimulation in naïve animals (Figure 2A). Subsequently, we moved these nicotine-treated animals to nicotine-free plates for a recovery period before subjecting them to another round of treatment. After three doses of such treatment, the same low concentration of nicotine then evoked robust locomotion-stimulation in these animals, indicating that worms can be sensitized to nicotine (Figure 2B and 2C).
It may be argued that the observed nicotine sensitization might be caused by the accumulation of nicotine in worms. Two observations argue against this possibility. First, nicotine sensitization can only be seen with intermittent nicotine treatments (Figure 2D), whereas continuous nicotine treatments, which would presumably result in the accumulation of more nicotine, failed to induce sensitization (Figure 2D). Second, the sensitization effect did not peak until the treated animals rested on nicotine-free plates for one hour after the last dose of nicotine treatment (Figure 2E), providing further evidence that the observed sensitization effect was not simply due to the accumulation of nicotine.
Taken together, our data show that worms exhibit nicotine-dependent behavior: they respond to acute nicotine treatment, develop tolerance to chronic nicotine exposure, display withdrawal symptoms upon nicotine cessation, and become sensitized to nicotine after repetitive nicotine challenges. These behavioral responses to nicotine seem to parallel those observed in vertebrates.
Having developed a model for characterizing nicotine-dependent behavior, we then asked what genes may underlie this behavior in C. elegans. In vertebrates, the psychostimulatory effects of nicotine require nicotinic acetylcholine receptors (nAChRs), the molecular target of nicotine (Champtiaux and Changeux, 2004). As a first step to test whether these receptors are also required for nicotine responses in C. elegans, we challenged worms with DHβE, a nAChR competitive antagonist (Champtiaux and Changeux, 2004). This antagonist suppressed the acute nicotine response as well as nicotine sensitization in wild-type animals (Figure 3A). In vertebrates, nAChR antagonists can mimic the effects of nicotine cessation by inducing withdrawal-like symptoms in nicotine-treated animals in the presence of nicotine (Kenny and Markou, 2001). We also observed a similar phenomenon in C. elegans induced by DHβE (Figure 3B). These results provide strong pharmacological evidence that nicotine-dependent behavior in C. elegans requires nAChRs.
We next sought to provide genetic evidence for the requirement of nAChRs for nicotine responses in C. elegans. The C. elegans genome encodes 28 nAChRs (Jones and Sattelle, 2004), most of which have mutants available (Figure 3C). We screened these mutants and found that acr-15 and acr-16 mutant worms lacked response to acute nicotine treatment (Figure 3D), though both mutants were otherwise superficially wild-type [(Francis et al., 2005; Touroutine et al., 2005) and Figure S1]. As a result of this defect, the mutant worms were also defective in nicotine withdrawal and sensitization (Figure S2A–B). Thus, ACR-15 and ACR-16 are required for nicotine-dependent behavior. unc-63 and unc-38 mutants were also defective in response to nicotine (Figure 3D); however, it remains possible that such a deficit might result from a nonspecific defect in locomotion because both mutants particularly unc-63 are severely uncoordinated (Culetto et al., 2004; Fleming et al., 1997). Nonetheless, our results demonstrate that nicotine-dependent behavior in C. elegans requires nAChRs.
The essential roles of nAChRs in nicotine-dependent behavior prompted us to explore the possibility that mammalian nAChRs may functionally substitute for C. elegans nAChRs in this behavior. As the mouse α4β2 heteromeric channel is the only nAChR that has thus far been found to be essential and sufficient for mediating nicotine dependence in mice (Champtiaux and Changeux, 2004; Maskos et al., 2005; Tapper et al., 2004), we expressed this mouse nAChR as a transgene in the acr-16 mutant background under the acr-16 promoter. We found that mouse α4β2 rescued the mutant phenotype in acute response, withdrawal and sensitization, though the transgene did not significantly affect locomotion of wild-type worms (Figure 3E, S2A–B and data not shown). Another mouse nAChR, α7, failed to rescue the mutant phenotype (Figure 3E). While it is possible that such a negative result might simply stem from aberrant expression of mouse α7 in worm cells, it is nonetheless noteworthy that knockout studies in mice do not support an essential role for α7 in nicotine dependence (Orr-Urtreger et al., 1997). The observation that mammalian nAChRs can functionally substitute for their C. elegans homologue in response to nicotine strongly suggests that at least some of the genes regulating nicotine responses in mammals are functionally conserved in C. elegans.
Both neuron- and muscle-type nAChRs are present in mammals; however, it is the neuronal, but not the muscular nAChRs, that primarily mediate the psychostimulatory effects of nicotine (Champtiaux and Changeux, 2004). We therefore wondered whether this is also the case with C. elegans nAChRs. However, acr-16 is not neuron-specific, has strong expression in muscle cells, and has been reported to function as a nAChR in the neuromusclular junction [(Ballivet et al., 1996; Francis et al., 2005; Touroutine et al., 2005) and Figure S3B]. Similarly, acr-15 is also expressed in both neurons and muscles (Figure S3A). In the nervous system, acr-15 can be found in interneurons (including command interneurons), motor neurons and pharyngeal neurons (Figure S3A). We generated transgenic animals expressing ACR-15 specifically in neurons or muscles in the acr-15 mutant background under a neuron- or muscle-specific promoter, respectively. The neuronal but not the muscular expression of ACR-15 rescued the nicotine defects in acr-15 mutant animals including acute response, withdrawal and sensitization (Figure 4A and Figure S2A–B). Similar results were also obtained with acr-16 (Figure 4A and Figure S2A–B). It should be noted that nicotine can affect muscle function under certain conditions. For example, high concentrations of nicotine paralyze worms by acting in the muscle (Gottschalk et al., 2005). Nevertheless, under our conditions, nAChRs seem to primarily act in neurons to mediate their function in nicotine responses.
Interestingly, expression of acr-15 under the glr-1 promoter rescued the nicotine phenotype in acr-15 mutants (Figure 4A). This promoter drives expression primarily in command interneurons, the central players in the locomotion circuitry, and a few other neurons (Hart et al., 1995; Maricq et al., 1995), suggesting that acr-15 may act in some of these neurons to mediate its function in nicotine-dependent behavior. The same promoter, however, did not significantly rescue the acr-16 mutant phenotype (Figure 4A), indicating the involvement of additional or different sets of neurons for acr-16 function in nicotine-dependent behavior. Thus, we chose to focus on acr-15 for further characterization.
The observation that the glr-1 promoter-driven acr-15 rescued the mutant phenotype suggests that command interneurons, the central players in the locomotion circuitry, may be important for mediating nicotine-dependent behavior. Therefore, we killed these neurons (AVA, AVB, AVD and PVC) with a laser. Worms lacking PVC or AVD did not exhibit a significant defect in response to nicotine incubation (Figure 4B). In contrast, ablation of AVA rendered worms unresponsive to nicotine, demonstrating an essential role for this neuron in nicotine-dependent behavior (Figure 4B). Ablation of AVB also impaired worms’ response to nicotine, though the ablated worms retained residual nicotine response (Figure 4B). However, unlike other command interneurons, AVB ablation makes worms severely uncoordinated [(Chalfie et al., 1985) and data not shown]; thus, it remains possible that the observed nicotine defects in these worms may result from a nonspecific deficit in locomotion. As such, we decided to focus on AVA for further characterization.
To provide physiological evidence that command interneurons are important for nicotine responses, we recorded the activity of these neurons by calcium imaging of live animals expressing G-CaMP, a genetically-encoded calcium sensor (Nakai et al., 2001). G-CaMP and cameleon have been successfully used in C. elegans to monitor neuronal activity (Kahn-Kirby et al., 2004; Li et al., 2006; Suzuki et al., 2003). DsRed was co-injected as a reference marker, allowing for ratiometric imaging. We focused on AVA because of its essential role in nicotine-dependent behavior and its expression of ACR-15 (Figure 4A and Figure S3A). In naïve animals, acute nicotine perfusion elicited robust calcium responses in AVA (Figure 4C and 4G). In contrast, such responses were greatly reduced in the animals that were behaviorally adapted to nicotine by chronic nicotine treatment (Figure 4D and 4G), though ACR-15 was up-regulated in these animals (Figure S3C–D). Chronic nicotine treatment also up-regulates nAChRs in mammals, the mechanism of which is not fully understood (Marks et al., 1986). Notably, in the animals that were behaviorally sensitized to nicotine, the nicotine-induced calcium responses were significantly potentiated (Figure 4E and 4G). Importantly, very little response to nicotine perfusion was observed in acr-15 mutant animals (Figure 4F and 4G), suggesting that ACR-15 is important for mediating the observed calcium responses. In support of this in vivo observation, we found that when expressed in HEK293T cells, ACR-15 was capable of forming a functional nAChR that can be activated by nicotine (Figure S4). Taken together, these results provide a cellular and molecular mechanism for nicotine regulation of locomotion behavior, and reveal a correlation between behavioral responses and cellular physiology.
Having examined some genes known to regulate nicotine responses, we then sought to identify novel genes involved in the process. In an effort to characterize C. elegans TRPC channels, we became intrigued by the possibility that these channels might regulate nicotine-dependent behavior. As a first step, we challenged wild-type animals with 2-APB, a TRPC channel inhibitor (Montell, 2005; Ramsey et al., 2006), and found that this drug abolished the response to acute nicotine treatment as well as to nicotine withdrawal in wild-type animals, suggesting that TRPC channels are required for nicotine-dependent behavior (Figure 5A and Figure S5A). However, 2-APB impinges on several other targets in addition to TRPC channels (Montell, 2005; Ramsey et al., 2006). Therefore, we decided to examine TRPC mutants.
The C. elegans genome encodes three TRPC channel homologues, TRP-1, TRP-2 and TRP-3 (Xu and Sternberg, 2003). They all share the same domain structure with human and fly TRPCs (Figure 5B). These include 3–4 ankyrin repeats and a coil-coil domain in the N-terminus followed by six putative transmembrane domains and a TRP domain in the C-terminal cytoplasmic tail (Figure 5B). We isolated one trp-1 allele that deleted the promoter region as well as the majority of the N-terminus, and one trp-2 allele that ablated half of the transmembrane domains (Figure 5C and 5D). Both alleles are likely to be null.
Both trp-1 and trp-2 mutant animals were superficially wild-type (Figure S1 and data not shown), though some moderate locomotion abnormalities can be detected with our tracking system (Feng and Xu, unpublished observations). Importantly, both trp mutants lacked response to acute nicotine treatment, consistent with our pharmacological results (Figure 5E). As a result of this defect, these mutants were also defective in nicotine withdrawal and sensitization (Figure S5A–B). As a control, mutants lacking the sperm-specific TRPC channel TRP-3, the TRPV channel OSM-9 or the TRPM channel GTL-1 all responded normally to nicotine incubation (Colbert et al., 1997; Teramoto et al., 2005; Xu and Sternberg, 2003) (Figure 5E). Transgenic expression of wild-type copies of the trp-2 gene in the trp-2 mutant background restored mutant animals’ responses to nicotine including acute response, withdrawal and sensitization (Figure 5E and Figure S5A–B). Similar results were obtained with trp-1 rescue (Figure 5E and Figure S5A–B). Therefore, TRPC channels are required for nicotine-dependent behavior in C. elegans.
TRP-1 has been reported to be expressed in multiple classes of neurons, such as interneurons (including command interneurons), motor neurons, pharyngeal neurons and sensory neurons (Colbert et al., 1997). TRP-2 is also expressed in these types of neurons (Figure S5D–E). Expression of TRP-2 under the glr-1 promoter can rescue the trp-2 mutant phenotype, including acute response, withdrawal and sensitization (Figure 5E and Figure S5A–B). Similar results were obtained for trp-1 (Figure 5E). This promoter was also able to rescue the acr-15 mutant phenotype (Figure 4A), suggesting that TRPC channels and ACR-15 might act in the same groups of neurons or circuits to regulate nicotine-dependent behavior.
We then asked whether worm TRPCs can function, like their mammalian counterparts, as receptor-operated channels (Montell, 2005; Ramsey et al., 2006). Although the precise mechanisms leading to activation of mammalian TRPCs remain uncertain, they all can be activated following the stimulation of phospolipase Cβ (PLCβ) when expressed in heterologous systems (Montell, 2005; Ramsey et al., 2006). We used TRP-2 as an example and isolated its cDNA by RT-PCR. Expression of TRP-2 in HEK293T cells promoted receptor-operated calcium entry elicited by perfusion of carbachol (Figure 6A, 6B and 6G). Carbachol is known to induce such calcium entry by stimulating PLCβ via its endogenous receptors in HEK293 cells that are coupled to heterotrimeric G-proteins (Montell, 2005; Ramsey et al., 2006). Further evidence came from the observation that the TRP-2-dependent activity in HEK293T cells can be blocked by U73122, a PLC inhibitor (Figure 6H) (Montell, 2005; Ramsey et al., 2006). As is the case with mammalian TRPCs, the TRP-2-dependent activity in HEK293T cells was sensitive to 2-APB (Figure 6H). In addition, TRP-2 appeared to be permeable to Ba2+ and Sr2+, a feature shared by several mammalian TRPCs (Figure 6C–6G) (Montell, 2005; Ramsey et al., 2006). These observations provide strong evidence that TRP-2 can function as a receptor-operated channel either by its own or by interacting with endogenous TRP proteins.
The requirement of PLCβ for TRP-2 activation in vitro raises the possibility that PLCβ may play a role in nicotine-dependent behavior in vivo. egl-8 encodes the worm PLCβ homolog that is ubiquitously expressed in the nervous system (Lackner et al., 1999; Miller et al., 1999). egl-8 mutant animals did not exhibit significant response to acute nicotine incubation or withdrawal (Figure 7A and Figure S6A). We also examined the role of Gq/11, because these proteins are known to function upstream of PLCβ (Montell, 2005). A reduction-of-function Gq/egl-30 mutant lacked response to nicotine incubation; however, we cannot exclude the possibility that such a defect might result from a nonspecific deficit in locomotion, because this mutant is severely uncoordinated (Brundage et al., 1996; Miller et al., 1999). Nonetheless, our results demonstrate an in vivo role for PLCβ in regulating nicotine-dependent behavior in C. elegans, and are also consistent with the role of TRPC channels in this behavior.
In light of the functional similarity between TRP-2 and mammalian TRPCs in heterologous systems, we reasoned that mammalian TRPCs might be able to substitute for the function of TRP-2 in nicotine-dependent behavior in C. elegans. To explore this possibility, we generated transgenic animals expressing human TRPC genes in the trp-2 mutant background under the trp-2 promoter. Three human TRPC genes (hTRPC1, 3 and 4) were tested, with each representing a subgroup of the human TRPC subfamily (Montell, 2005; Ramsey et al., 2006). While negative results were obtained with hTRPC1 and 4, expression of hTRPC3 restored the responses of trp-2 mutant animals to nicotine including acute response, withdrawal and sensitization (Figure 7A and Figure S6). These results reveal functional conservation of TRPC channels in regulating nicotine-dependent behavior.
The question arises as to how TRPC channels regulate nicotine-dependent behavior. One possibility would be that nAChRs may depend on TRPC channels for their expression. However, this does not seem to be case. For example, TRP-2 was not required for ACR-15 expression (Figure S3E and data not shown). As TRPCs and ACR-15 may act in the same groups of neurons or circuits, we tested whether TRPC channels can functionally regulate nAChR activity by imaging nicotine-induced calcium responses in live animals expressing the genetically-encoded calcium sensor G-CaMP. We found that nicotine-induced calcium responses in the command interneuron AVA were greatly reduced in trp-2 and trp-1 mutant worms (Figure 7B–E). Thus, it appears that TRPC channels can functionally regulate nicotine-induced neuronal activity in the locomotion circuitry, providing a cellular mechanism for TRPC channel regulation of nicotine-dependent behavior.
In the current study, we developed a C. elegans model of nicotine-dependent behavior. Our results indicate that C. elegans displays several types of behavioral responses to nicotine that parallel those observed in vertebrates. These include acute response, tolerance, withdrawal and sensitization. In addition, we show that nicotine responses in C. elegans require nAChRs, the molecular targets of nicotine that are known to mediate nicotine dependence in mammals (Champtiaux and Changeux, 2004; Laviolette and van der Kooy, 2004). These results demonstrate that at least some of the genes regulating nicotine responses in mammals are functionally conserved in C. elegans, and suggest that C. elegans is a valuable system for characterizing genes regulating nicotine responses.
While only 17 nAChR genes are present in mammals, the C. elegans genome encodes 28 such genes. Among the worm nAChR genes that have been examined for expression patterns, all have been found to be expressed in neurons (Jones and Sattelle, 2004). Although much is known about the role of these nAChRs in regulating muscle activity (Francis et al., 2005; Gottschalk et al., 2005; Richmond and Jorgensen, 1999; Touroutine et al., 2005; Waggoner et al., 2000), their functions in the nervous system are not well understood. We have shown that at least two nAChR genes, acr-15 and acr-16, are required for nicotine-dependent behavior. Both nAChRs can be activated by nicotine when expressed in heterologous systems [Figure S4 and (Ballivet et al., 1996)]. Although these two nAChRs are expressed in both neurons and muscles, as is the case with their mammalian counterparts, we have found that under our conditions they primarily act in neurons to regulate nicotine-dependent behavior. One site of action for ACR-15 seems to be in the command interneurons. Nevertheless, it remains unclear in which neurons ACR-16 acts to mediate its function in nicotine responses. Nicotine dependence in mammals is a highly complex phenomenon entailing the function and coordination of multiple nAChR genes and brain regions. In worms, it may also involve the action of multiple nAChRs expressed in different classes of neurons that are directly or indirectly connected to the locomotion circuitry.
Our laser ablation and calcium imaging experiments suggest that command interneurons are important for nicotine-dependent behavior. This is consistent with the critical role of these neurons in locomotion (Chalfie, 1988). Nevertheless, our results do not exclude the involvement of other types of neurons in regulating nicotine-dependent behavior. Among the four major pairs of command interneurons, AVA neurons are essential for nicotine-dependent behavior; AVB neurons may also play an important role. One function for AVA neurons is to regulate spontaneous reversal frequency during locomotion (Chalfie et al., 1985; Zheng et al., 1999). However, these neurons also receive synaptic input from as well as synapse onto other command interneurons including AVB and PVC, both of which are known to regulate forward movement (Chalfie et al., 1985; White et al., 1986). Upon nicotine stimulation, AVA and AVB may tune the activity of the locomotion circuitry, leading to the observed behavioral effects.
We have also begun to identify novel proteins regulating nicotine-dependent behavior in C. elegans and found that TRPC channels are required for this behavior. Specifically, trp mutant worms are defective in the acute nicotine response, which might lead to defects of these mutants in other nicotine responses such as nicotine withdrawal and sensitization. The role of TRPC channels in nicotine responses is also supported by the observation that PLCβ, a protein important for TRPC channel activation, is critical for nicotine-dependent behavior in C. elegans.
How do TRPC channels regulate nicotine-dependent behavior? Our rescuing experiments suggest that TRPC channels and the nAChR ACR-15 may act in the same groups of neurons or circuits to mediate their function in nicotine responses. Indeed, worms lacking TRPC channels are defective in nicotine-induced, ACR-15-dependent calcium responses in some command interneurons. Thus, while other mechanisms may also contribute, one mechanism by which TRPC channels regulate nicotine-dependent behavior appears to be through functionally modulating nicotine-induced command interneuron activity in the locomotion circuitry. TRPC channels may do so by acting directly in command interneurons and/or indirectly via network activity.
All six human TRPC channels are expressed in the central nervous system (CNS) (Montell, 2005; Ramsey et al., 2006). Despite extensive in vitro studies in cell culture systems implicating mammalian TRPC channels in various neuronal activities in the CNS, such as synaptic transmission, growth cone guidance and morphology, and neurite extension [reviewed in (Ramsey et al., 2006)], the genetic evidence supporting such roles for these channels is still lacking. Our studies identify a novel role for TRPCs in nervous system function, and reveal an unexpected functional link between TRP family channels and nicotinic signaling. The observation that a human TRPC gene can functionally substitute for a C. elegans TRPC channel also raises the possibility that these channels might regulate nicotine dependence and perhaps other types of substance dependence in mammals.
The following mutant alleles were used in the study: acr-5(ok180), acr-8 (ok1240), acr-9(ok933), acr-11(ok1345), acr-12(ok367), acr-14(ok1155), acr-15(ok1214), acr-16(ok789), acr-18(ok1285), acr-19(ok967), acr-21(ok1314), deg-2(u695)deg-3(u662), deg-3(tu1851), eat-2(ad465), eat-4(ky5), egl-8(n488), egl-30(md186), gtl-1(ok375), lev-1(e211), lev-8(x15), osm-9(ky10), trp-1(sy690), trp-2(sy691), trp-3(sy693), unc-29(x29), unc-38 (x20), and unc-63(x13).
To generate the unc-119 promoter-driven transgenic lines, the unc-119 promoter was first amplified by PCR from a plasmid (PBY103), fused by PCR with a PCR fragment (amplified from genomic DNA) encoding the coding region of acr-15 and acr-16, and injected into the acr-15(ok1214) and acr-16(ok789) mutant background, respectively. Transgenic lines expressing the myo-3 and glr-1 promoter-driven transgenes were generated with the same strategy. The myo-3 promoter and glr-1 promoter was amplified by PCR from pPD136.64 (A. Fire) and genomic DNA, respectively (Hart et al., 1995; Maricq et al., 1995). To make transgenic worms expressing the transgene Pacr-16::mouse α4β2, the acr-16 promoter (~6 kb) was amplified by PCR from genomic DNA, fused by PCR with a PCR fragment of the mouse α4 cDNA orβ2 cDNA coding region linked to unc-54 3′ UTR, respectively, and co-injected at a ratio of 2:3 into the acr-16(ok789) mutant background. To generate transgenic lines expressing Ptrp-2::hTRPC3, the trp-2 promoter (~3.5 kb) was amplified from the cosmid R06B10, fused by PCR with a PCR fragment of human TRPC3 cDNA coding region linked to the unc-54 3′ UTR, and directly injected into the trp-2(sy691) mutant background. The Ptrp-2::hTRPC1 and Ptrp-2::hTRPC4 transgenes were constructed with the same strategy. The cosmids ZC21 and R06B10 were used to rescue the trp-1(sy690) and trp-2(sy691) mutants, respectively. At least two independent lines were analyzed for each transgene. Both trp alleles were backcrossed to N2 for seven times before behavioural analysis.
L4 hermaphrodites were picked 16 hours before behavioral analysis. The NGM plates used for tracking were freshly spread with a thin layer of E. coli OP50 five minutes prior to tracking. Tracking was performed at 20–21°C and at a relative humidity of ~40% with the lid off. The tracking system consists of a stereomicroscope (Zeiss Stemi 2000C) mounted with a digital camera (Cohu 7800), a digital motion system (Parker Automation) that follows worm movement, and a home-developed software package. To record locomotion, animals’ images were grabbed at a rate of 2 Hz for 16 mins. The locomotion velocity of the animal at each time point, computed as centroid displacement (mm) per second, was plotted and displayed in real time during tracking. The vision/motion data were also compressed, integrated and stored as a commonly used multimedia file format (AVI). Nicotine was included in media right before plates were poured. DHβE and 2-APB were directly spread on the surface of NGM plates and allowed to diffuse for >16 hours prior to use. Animals were pre-incubated with DHβE for 1 hour prior to analyzing their response to nicotine.
Methods for quantifying locomotion-stimulation are described in the Supplemental Data.
We thank William Schafer, Gary Schindelman, Patrick Hu, Raad Nashmi and Craig Montell for comments and advice, Junichi Nakai for the G-CaMP plasmid, Millet Treinin for the deg-3 strain, Henry Lester for mouse nAChR cDNA plasmids, and Barbara Perry and Rahul Mahapatra for technical assistance. Z.F. was inspired to study nicotine by previous training with W.S. Some strains were obtained from the CGC and C. elegans Gene Knockout Consortium. The work was supported by the USPHS training grant T32EY017878 (A.W.) and T32GM008322 (B.J.P.), HHMI with which P.W.S. is an investigator, NIDA (7R01DA018341-02 to P.W.S.), Univ. of Michigan BSSP program (X.Z.S.X), and grants from the American Legacy Foundation via UMTRN (X.Z.S.X) and NIGMS (X.Z.S.X.).
Author contributionsZF and XZSX conceived and designed the experiments. ZF and WL performed the experiments and analyzed the data. AW, BJP and ERL helped perform some experiments and paper writing. PWS contributed critical reagents, intellectual input and help with paper writing. XZSX and ZF wrote the paper.
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