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Chronic administration of antipsychotic drugs produces adaptive responses at the cellular and molecular levels that may be responsible for both the main therapeutic effects and rebound psychosis, which is often observed upon discontinuation of these drugs. Here we show that some antipsychotic drugs produce significant functional changes in serotonergic neurons that directly impact feeding behavior in the model organism, Caenorhabditis elegans. In particular, antipsychotic drugs acutely suppress pharyngeal pumping, which is regulated by serotonin from the NSM neurons. By contrast, withdrawal from food and drug is accompanied by a striking recovery and overshoot in the rate of pharyngeal pumping. This rebound response is absent or diminished in mutant strains that lack tryptophan hydroxylase (TPH-1) or the serotonin receptors SER-7 and SER-1, and is blocked by serotonin antagonists, which implicates serotonergic mechanisms in this adaptive response. Consistent with this, continuous drug exposure stimulates an increase in serotonin and the number of varicosities along the NSM processes. Cyclosporin A and calcineurin mutant strains mimic the effects of the antipsychotic drugs and reveal a potential role for the calmodulin-calcineurin signaling pathway in the response of serotonergic neurons. Similar molecular and cellular changes may contribute to the long-term adaptive response to antipsychotic drugs in patients.
Antipsychotic drugs rapidly bind to and block dopamine and serotonin receptors in the brain. Receptor occupancy occurs within hours (Coppens et al., 1991), yet significant clinical improvement and the emergence of adverse side effects may require weeks or even months to manifest (Lieberman et al., 1993; Conley et al., 1997). The delay in onset of some biological effects of the antipsychotic drugs may reflect the time required for molecular and cellular adaptation to continuous exposure to drug, and the establishment of a new homeostatic state (Hyman and Nestler, 1996). Studies of the long-term effects of antipsychotic drugs in humans and rodents reveal changes in the expression of dopamine and serotonin receptors in different brain regions (Silvestri et al., 2000; Tarazi et al., 2002), which may affect behavior. However, the functional response of the nervous system to these drugs has not been analyzed simultaneously at the molecular, cellular, and behavioral levels.
The model organism, C. elegans, has provided important molecular insights into the role of Ca2+-signaling pathways in behavioral adaptation to neuroactive drugs (Schafer and Kenyon, 1995; Feng et al., 2006). Antipsychotic drugs produce significant changes in C. elegans behavior, including egg-laying, locomotion and foraging (Weinshenker et al., 1995; Donohoe et al., 2006, 2008). The main goal of the present study was to take advantage of this model system to characterize the cellular and molecular response to antipsychotic drugs in relation to changes in feeding behavior (pharyngeal pumping) mediated via a well-defined neural circuit.
Pharyngeal pumping in C. elegans is controlled by the pharyngeal nervous system, in particular the MC, M3, and NSM neurons (Raizen et al., 1995; Niacaris and Avery, 2003). Serotonin stimulates pumping through a complex response that involves several receptor subtypes, including cholinergic and glutamatergic, but not dopaminergic receptors (Niacaris and Avery, 2003; Hobson et al., 2006). The tph-1(mg280) mutant strain, which lacks serotonin, pumps more slowly (Sze et al., 2000) and shows greater variability in the rate of pharyngeal pumping (Hobson et al., 2006). This is consistent with serotonin providing homeostatic regulation by establishing the dynamic range of a behavior (Azmitia, 2007).
Antipsychotic drugs block serotonin receptors (Meltzer, 1999), and affect signaling pathways that regulate tryptophan hydroxylase (TPH) (Mockus and Vrana, 1998; Ninan et al., 2003; Estevez et al., 2004; Zhang et al., 2004; Donohoe et al., 2008), the rate-limiting enzyme in serotonin biosynthesis. Therefore, it would not be surprising if these drugs produced functional changes in serotonergic systems of C. elegans, which could alter behavior such as pharyngeal pumping. As reported here, first-generation antipsychotics, trifluoperazine and fluphenazine, the atypical (second-generation) drug, clozapine, and the calcineurin inhibitor, cyclosporin A, increased the expression of TPH-1::green fluorescent protein (TPH-1::GFP) and the number of varicosities along NSM neurites, with accompanying adaptive changes in pharyngeal pumping, which is regulated by these neurons. Mechanistic studies of this response implicate Ca2+-calmodulin-calcineurin signaling in the regulation of long-term functional changes in serotonergic neurons that affect this behavior.
Trifluoperazine, fluphenazine, clozapine, methiothepin, cyclosporin A, and d-tubocurarine were purchased from Sigma (St. Louis, MO). Calmidazolium and cycloheximide were purchased from Calbiochem (San Diego, CA). Olanzapine was a gift from Eli Lilly and Co. (Indianapolis, IN).
C. elegans strains were grown and maintained as described by Brenner (1974) at 20°C with E. coli strain OP50 as the food source. The N2 var. Bristol strain served as the wild-type reference. The mutant strains used for these studies were: GR1321 tph-1(mg280), GR1366 tph-1::GFP, DA1814 ser-1(ok345), DA2109 ser-1(ok345);ser-7(tm1325), DA2100 ser-7(tm1325), AQ866 ser-4(ok512), JY190 osm-9(y26), CB1416 unc-86(e1416), PR675 tax-6(p675), and KJ300 cnb-1(jh103). The strains were obtained from the Caenorhabditis Genetics Center (CGC, Minneapolis, MN) or were kindly provided by Dr. Leon Avery in the case of the ser-7 and ser-1;ser-7 strains.
Drug plates were made as previously described (Donohoe et al., 2006). Drugs were dissolved in DMSO (trifluoperazine, fluphenazine, clozapine, olanzapine, methiothepin), ethanol (calmidazolium, cyclosporin A), or dilute acetic acid, (curare) for the stock solution. Drug plates were allowed to dry overnight and were used the next day. Unseeded plates contained no bacteria or peptone.
The rate of pharyngeal pumping was scored by counting the number of pharyngeal bulb contractions over 30 sec according to the methods of Sawin et al. (2000). Pumping rate was measured in animals (40 per group unless specified) (1) on food and drug, or (2) on unseeded plates 6 or 24 h after removal from food. To determine the acute effects of drugs on pharyngeal pumping (corresponding to Fig. 1A), fed adult N2 animals were transferred directly onto plates with either drug or solvent (control) and were then scored at various times afterward (5–440 min). For the longer-term studies (depicted in Figs. 1B, ,2,2, ,33 and and6),6), animals were exposed to solvent or drug on plates from hatching and were maintained on these plates until the population reached the fourth larval stage. Standardization to the L4 stage rather than the absolute time of incubation was necessary because the highest concentrations of certain drugs (e.g., trifluoperazine and calmidazolium at 80 μM) slow larval maturation. At the L4 stage, pharyngeal pumping was measured in a total of 40 animals grown on plates seeded with bacteria and containing solvent or drug. Animals were then collected from the solvent/drug plates, washed 3X with M9 buffer, and were transferred to plates that contained no food or drug. Pharyngeal pumping rate was scored 6 and/or 24 h after the animals were withdrawn from both food and drug. Pilot studies showed that the drugs effectively cleared from animals after a 4–5 h washout period as judged by the time required to recover normal pumping rates following suppression of pumping caused by acute (30–60 min) incubation with drugs. After the 4–5 h washout step, pharyngeal pumping does not exceed the normal rate unless there has been a prolonged incubation with drug for 6 h or more.
For experiments with methiothepin or curare (shown in Figs. 3 & 6), animals were incubated on plates with solvent or drug as before, until they reached the L4 stage. Next, they were transferred to plates that contained methiothepin (100 μM) or curare (5 mM), but no food or other drug. After an additional 24 h on these plates, we measured pharyngeal pumping. For the kinetic experiments (depicted in Fig. 1C), animals were grown until the late L2-early L3 larval stage, and some were transferred to seeded plates with drug for 0.5, 3, 6, 12, and 24 h. Control animals (no drug) and animals exposed to drug for the various times were then transferred off food and drug for 24 h prior to evaluating pharyngeal pumping and the rebound response.
Animals carrying the tph-1::gfp fusion construct (Sze et al., 2000) were exposed from hatching until the fourth larval stage on plates that contained either solvent (control) or drug. At this time, the animals were washed off the plates with M9 buffer (containing 50 mM sodium azide) and were placed on microscope slides for fluorescence analysis. Total fluorescence in the NSM processes was measured as described elsewhere (Donohoe et al., 2008) and the number of varicosities (thickenings > 2 times the diameter of the process) was also determined. Digital photomicrographs were taken with a CoolSNAP monochrome camera attached to a Nikon Eclipse TE300 inverted microscope equipped with an epifluorescence attachment (including a green fluorescent protein filter), and Nomarski optics. The number of varicosities along the pharyngeal nerve ring of the NSM processes was counted in control and drug-treated animals. Total fluorescence along the NSM processes (integrated density) was calculated with Image J (NIH; Bethesda, MD). Total fluorescence represents the integrated density (mean fluorescence multiplied by the total area) set at a threshold 4 standard deviations above the background. Background was calculated by obtaining measurements from a non-fluorescent area adjacent to the NSM processes. The region of interest was defined by circumscribing the individual processes extended from the NSM neurons.
Serotonin immunostaining was performed according to Zhang et al. (2005). Animals were stained for identical time intervals with a rat anti-serotonin monoclonal antibody (Chemicon, Temecula, CA; MAB352) and with a goat anti-rat IgG conjugated to Alexafluor 568 (Molecular Probes, Eugene, OR; A11077).
For the cycloheximide experiments, a synchronized population of tph-1::gfp animals (at the L4 larval stage) was divided into four groups and placed on seeded plates containing: (1) solvent (control), (2) 40 μM trifluoperazine, (3) 7.5 mM cycloheximide, and (4) 7.5 mM cycloheximide + 40 μM trifluoperazine. Mean fluorescence in the NSM cell bodies of animals was measured 12 h and 24 h after the animals were transferred to experimental plates. The animals remained fully viable over this time as judged by their normal response to harsh touch.
A two-tailed unpaired Student’s t-test was used to analyze statistical differences between groups in pharyngeal pumping experiments. Data from all other quantitative experiments were analyzed by a one-way ANOVA with Tukey post hoc analysis. Actual P values are included in the text, and in the figures significant differences are indicated by asterisks: *p< 0.05, **p< 0.01.
Initially, we characterized the acute effect of antipsychotic drugs on the rate of pharyngeal pumping by exposing wild-type (N2) and mutant strains for various times to concentrations of drug previously shown to block dopamine-mediated behavior in C. elegans (Donohoe et al., 2006). In N2 animals, the pharyngeal pumping rate is ~200–220 pumps/min on food, and decreases to ~120 pumps/min when food is absent. The effect of various antipsychotic drugs on pharyngeal pumping is shown in Fig. 1. At the 0-min time point, the pumping rate is the same as on bacteria. As seen in Fig. 1A, the first generation antipsychotic drugs, trifluoperazine and fluphenazine, produced rapid onset suppression of pharyngeal pumping in well-fed N2 animals, whereas the second-generation drugs, clozapine and olanzapine, produced a modest, but significant suppression to around 170 pumps/min after 160 min exposure. The acute onset of suppression observed with trifluoperazine and fluphenazine was consistent with the ability of these drugs to inhibit nicotinic cholinergic receptors (Connolly et al., 1992), which mediate significant aspects of pharyngeal pumping (Raizen et al., 1995).
Pharyngeal pumping was suppressed as long as animals were exposed to antipsychotic drugs, but recovered dramatically upon drug withdrawal. Initial studies revealed that when animals were transferred from seeded plates with fluphenazine to seeded plates without drug, the rate of pharyngeal pumping rebounded over 48 h and rose to an average of 264±5 pumps per minute, which is significantly above the normal pumping rate on food, 224±4 (p < 0.01; n=20). This rebound response (recovery plus overshoot) approaches the maximum pumping rate for C. elegans and imposes a ceiling effect on studies of this phenomenon. Thus, the presence of bacteria affects pumping in concert with any effects of the drug. By comparison, the rate of pharyngeal pumping is well below maximum levels after short periods of food deprivation. Therefore, we chose to study the rebound response to drugs in animals deprived of food for 6–24 h. It is known that L4-young adult N2 animals survive long periods of starvation (> 3 d) with little adverse effect (Weinkove et al., 2006; You et al., 2006).
From these observations, we derived the optimum conditions for studying the rebound response to drugs, namely exposure to drug on food for 2–3 d followed by removal from both food and drug for 6–24 h before measuring pharyngeal pumping. Consequently, wild-type N2 animals were incubated from hatching until the L4 stage with trifluoperazine, fluphenazine, clozapine, and olanzapine, and were then removed from food and drug for 24 h for testing. Animals previously exposed to trifluoperazine, fluphenazine and clozapine displayed a significant rebound response in pharyngeal pumping (Fig. 1B), which remained above control levels for more than 72 h (data not shown). Therefore, the rebound effect of drug is also observed when drug withdrawal is coupled to a change in food state. Olanzapine induced a slight rebound in pharyngeal pumping, but this response was not statistically significant (P = 0.08).
To explore the kinetics of the rebound response, we placed a synchronized population of N2 animals (L3 larval stage) on plates containing trifluoperazine (40 μM) and Escherichia coli (strain OP50) as food for fixed time intervals (0.5, 3, 6, and 24 h). We then transferred the animals off food and drug for 24 h, and again measured their pumping rate. A significant overshoot in pharyngeal pumping was observed only in animals exposed to trifluoperazine for 6 h or longer; the response was even higher after 24 h of continuous drug exposure than after a 6-h exposure (Fig. 1C).
Pharyngeal pumping is partially controlled by a neural circuit that involves serotonergic modulation of the pharyngeal motor neurons, MC and M3, which release acetylcholine and glutamate, respectively, directly onto pharyngeal muscle (Niacaris and Avery, 2003) (see Fig. 2A). Because serotonin is necessary to sustain high rates of pumping in C. elegans (Sze et al., 2000; Hobson et al., 2006), we tested whether the antipsychotic drugs clozapine, fluphenazine, and trifluoperazine produced a rebound response in a mutant strain lacking serotonin due to a predicted null mutation in the gene that encodes tryptophan hydroxylase (tph-1) (Sze et al., 2000). The tph-1(mg280) strain shows a slight, but significant, reduction in the basal rate of pharyngeal pumping on food as compared to controls (Sze et al, 2000; and compare Fig. 2B with Fig. 1B & C). Nevertheless, tph-1 animals did not show an overshoot in pharyngeal pumping following drug withdrawal (Fig. 2B). Consistent with this finding, N2 animals treated with trifluoperazine for 24 h, and then transferred onto bacteria-free plates containing the selective serotonergic antagonist, methiothepin (100 μM), did not exhibit a rebound in pharyngeal pumping (Fig. 3A). Thus, serotonin was required for the rebound response to drug. The cholinergic antagonist, curare, partially blocked the overshoot in pharyngeal pumping (Fig. 3B), which showed that cholinergic signaling also played a role in the overall response. These data suggest that the rebound response is mediated, at least in part, through serotonergic modulation of the cholinergic MC neurons.
Only two pairs of serotonergic neurons, the NSM and the ADF neurons, are located in the vicinity of the pharyngeal neural circuit, and only the NSMs are located in the pharynx. The NSM neurons fail to produce serotonin in unc-86(e1416) animals (Sze et al., 2002), whereas the ADF neurons are largely devoid of serotonin in osm-9(y26) animals, which have a loss-of-function mutation in the TRPV channel gene (Zhang et al., 2004). Therefore, we tested unc-86(e1416) and osm-9(y26) mutants for the rebound response after chronic exposure to trifluoperazine (40 μM). Trifluoperazine produced a significant rebound in the osm-9 mutants, but not in the unc-86 mutants (Fig. 3C), which suggests that the NSM neurons are the major source of the serotonin that mediated the rebound response. Effects of the unc-86 mutation on other aspects of the response are not excluded, although basal pumping behavior in this strain is normal both on and off food.
To identify the serotonergic receptor(s) involved in the adaptive response to antipsychotic drugs, we examined trifluoperazine-induced rebound in mutant strains lacking the serotonin receptor genes ser-1(ok345), ser-4(ok512), ser-7(tm1325) and both ser-1(ok345) and ser-7(tm1325). A 6-h time point was chosen for these studies because some of the serotonin receptor loss-of-function strains increase basal pharyngeal pumping rates off food over 24–48 h. Comparisons between the N2 controls off food and the various mutant strains evaluated off food and trifluoperazine revealed a significant rebound response in the N2 positive controls and ser-4 animals, but not in the ser-7 strains as shown in Fig. 3D. Although ser-1 animals showed a significant rebound relative to controls, this response was greatly reduced in comparison to that observed in the wild-type animals. Thus, SER-7 (5-HT7 receptor of C. elegans) and SER-1 (5-HT2 receptor), but not SER-4 (5-HT1 receptor), are involved in the rebound response to antipsychotic drugs.
To further explore the relationship between serotonin production in the NSM neurons and antipsychotic drug exposure, we measured TPH-1::GFP levels in animals chronically exposed to antipsychotic drugs. Each NSM neuron has a process that bifurcates into a branch that crosses over to the opposite side and proceeds posteriorly along the dorsal nerve cord of the isthmus, and another ipsilateral branch that runs posteriorly along the subventral nerve cord of the isthmus (Fig. 4A). Varicosities along neuronal processes represent areas of neurotransmitter release (Bunin and Wightman, 1999; Nonet, 1999). Animals exposed to trifluoperazine (40 μM), fluphenazine (80 μM), and clozapine (160 μM) showed a significant increase in the number and size of varicosities around the pharyngeal nerve ring segment of the NSM processes (Fig. 4B, Fig. 5A), and in the amount of TPH-1::GFP fluorescence along the subventral and dorsal processes (Fig. 4B, Fig. 5B). This increase was not observed in tph-1::GFP animals chronically exposed to olanzapine (320 μM), which is consistent with the failure of olanzapine to elicit a rebound response. In addition, trifluoperazine (40 μM), and fluphenazine (80 μM) exposure caused a significant increase in serotonin levels as judged by immunofluorescence staining (Fig. 4C).
The Ca2+-calmodulin pathway, which is a secondary target of many antipsychotic drugs, regulates the activity and degradation of tryptophan hydroxylase (Mockus and Vrana, 1998). Phosphorylation of tryptophan hydroxylase prevents its degradation in the proteosome (Furukawa et al., 1993). Human TPH-1 is phosphorylated on Ser58 (Kumer et al., 1997), and the C. elegans tph-1::gfp construct (Sze et al., 2000) used in these studies has a similar consensus site near the N-terminus. Calcineurin, a calmodulin-dependent protein phosphatase, regulates the turnover and trafficking of proteins, including tyrosine hydroxylase (Cheramy et al., 1994). To test whether the antipsychotic drugs affect TPH-1 protein levels and pharyngeal pumping via calmodulin signaling, we evaluated the role of the Ca2+-calmodulin-calcineurin pathway in the rebound response. Calmidazolium (40 μM) is a selective calmodulin antagonist that is structurally and pharmacologically distinct from the antipsychotics. We exposed N2 animals to calmidazolium (40 μM) or the calcineurin inhibitor, cyclosporin A (20 and 40 μM), for 3 d, and then withdrew the animals from the compounds and food for 24 h to determine if either compound produced a rebound response. Significant rebounds in pharyngeal pumping were produced by both calmidazolium and cyclosporin A (Fig. 6A). Next, we examined whether calmidazolium and cyclosporin A enhanced the expression of TPH-1::GFP in the NSM neurons. Significant increases in TPH-1::GFP (Fig. 6B) and the number of varicosities along the nerve ring (Fig. 5C) were elicited by both calmidazolium and cyclosporin A. There was also an increase in the total amount of TPH-1::GFP fluorescence measured along the NSM processes (Fig. 5D). To relate the changes in TPH-1::GFP fluorescence to behavior, we next examined whether the potentiation of pharyngeal pumping upon withdrawal from cyclosporin A was abolished by inhibition of serotonergic function with methiothepin. For these experiments, animals were grown from hatching until the L4-young adult stage on cyclosporin A (20 or 40 μM) and were then removed from food and drug and transferred to plates with solvent (control) or methiothepin (100 μM). After 6 h and 24 h on the new plates, we measured pharyngeal pumping. As seen in Fig. 6C, cyclosporin A produced a small, but significant decrease in pharyngeal pumping on food. Following removal from food and drug, there was a significant overshoot in pharyngeal pumping at both the 6 h and 24 h time points. This potentiation was blocked by methiothepin, which implicates serotonin in the rebound response to cyclosporin A.
If inhibition of calcineurin is associated with an increase in serotonergic function of NSM neurons, we would also expect to observe changes in the behavior of strains lacking functional activity of this phosphatase. Therefore, we evaluated pharyngeal pumping in strains with loss of function in the regulatory, cnb-1(jh103), or catalytic subunits, tax-6(p675), of calcineurin. There was a significant increase in the rate of pharyngeal pumping in both of these strains 24 h after removal from food (Fig. 6D). In addition, we observed a significant increase in serotonin immunofluorescence in NSM neurons of tax-6 animals (Fig. 6E). A modest increase in varicosities with serotonin staining was observed in tax-6 animals, but this was not quantified. However, the total fluorescence data confirm higher levels of serotonin in calcineurin-deficient animals, which is consistent with our findings with the calcineurin inhibitors. Thus, the calmodulin-calcineurin pathway provides important regulation of TPH-1 and serotonin production in NSM neurons.
If the antipsychotic drugs indirectly inhibit calcineurin, there should be an increase in phosphorylated TPH-1, which can then interact with 14-3-3 proteins to evade protein degradation. Consequently, we should observe a slower rate of protein turnover in animals exposed to these drugs. To test this hypothesis, we analyzed the turnover rate of the TPH-1::GFP construct in control animals and animals exposed to trifluoperazine. Cycloheximide was used to block new protein synthesis, which allowed us to focus on the rate of loss (turnover) of existing TPH-1::GFP. L4-young adult stage tph-1::gfp animals (25 per group) were grown on seeded NGM plates in the presence of cycloheximide (7.5 mM) or in the presence of both cycloheximide (7.5 mM) and trifluoperazine (40 μM). For comparison, control animals (Con) were grown on seeded NGM plates with solvent, but no cycloheximide. We then measured the mean fluorescence in the NSM neurons 12 and 24 h later. In the absence of cycloheximide, there was stable expression of TPH-1::GFP over 24 h (Fig. 7), whereas expression levels steadily declined in its presence to near background levels. Trifluoperazine (40 μM) significantly slowed the loss of TPH-1::GFP signal, which is consistent with the idea that the stability of the enzyme is enhanced under these conditions. The stability of the full-length enzyme minus GFP may be different from that measured here. Nevertheless, these data provide initial support for the possibility that antipsychotic drugs increase TPH-1 levels, in part, by decreasing TPH-1 turnover, thereby increasing its accumulation over time. This interpretation does not exclude the possibility that the drugs also induce a concomitant increase in tph-1 gene expression.
This is the first report that antipsychotic drugs and calcineurin inhibitors produce a rebound response in behavior upon drug withdrawal that is mediated via changes in the levels of TPH-1 and serotonin in serotonergic neurons. Acutely, the drugs suppress pharyngeal pumping, whereas chronic exposure (> 6 h) followed by removal from drug and food is accompanied by recovery and overshoot in the pumping rate to levels that can far exceed control rates. We also report on the characterization of the molecular and cellular changes that give rise to this behavioral response. We show that the magnitude of the rebound in pharyngeal pumping is correlated with the amount of TPH-1::GFP and serotonin in the NSM neurons. Moreover, decreased signaling through the calmodulin-calcineurin pathway mimics the effects of the antipsychotic drugs, suggesting that this pathway plays a critical role. These data support previous studies in rats that found long-term administration of antipsychotic drugs increased extracellular serotonin in certain brain regions (Rastogi et al., 1981), however, unlike this study the effects on TPH were not investigated.
The role of serotonin in the rebound response to antipsychotic drugs was revealed by several independent observations. First, the response was abolished in mutant strains that completely lacked serotonin (tph-1) or failed to produce serotonin in the NSM neurons (unc-86). Second, the rebound and overshoot in pharyngeal pumping induced by both antipsychotic drugs and cyclosporin A was blocked with methiothepin, a selective antagonist of serotonin receptors in C. elegans. Third, there was a correlation between increased expression of TPH-1::GFP in the NSM neurons and the elevated rate of pharyngeal pumping. Acetylcholine appeared to play a secondary role because the rebound response was attenuated by the cholinergic antagonist, curare. However, the release of acetylcholine by the MC neuron is controlled by serotonin (Niacaris and Avery, 2003). Moreover, SER-1 receptors are expressed on pharyngeal muscle cells, which would allow direct modulation of pumping by the NSM neurons. Together, these findings suggest a dominant role for serotonin in the rebound response to antipsychotic drugs.
The recovery and overshoot in pharyngeal pumping after withdrawal from drug is mediated by SER-7 (5-HT7) and SER-1 (5-HT2) receptors. By contrast, SER-4 (5-HT1) receptors do not appear to contribute to the response. Our data agree closely with those of Hobson et al. (2006) who showed that SER-7 is the main receptor responsible for increased pharyngeal pumping in response to serotonin, and Dernovici et al. (2007) who reported a smaller, but significant contribution by SER-1. The fact that the overshoot in pharyngeal pumping upon drug withdrawal lasted for several days attests to the enduring nature of this adaptation.
Previous studies have established that Ca2+-signaling pathways govern adaptation to serotonin, dopamine, and nicotine (Schafer and Kenyon, 1995; Feng et al., 2006). In our system, converging evidence suggests that functional changes in the NSM neurons are mediated, in part, through Ca2+-signaling via calcineurin and calmodulin, the latter representing a secondary target of some antipsychotic drugs (Weiss et al., 1983). Trifluoperazine and fluphenazine have been shown to inhibit calmodulin-calcineurin activity in species such as Paramecium tetraurelia (Kissmehl et al., 1997), Leishmania donovani (Banerjee et al., 1999), and Candida albicans (Sanglard et al., 2003) that are evolutionarily more distant from mammals than C. elegans. Furthermore, the data are consistent with previous observations that these drugs inhibit C. elegans growth and development in relation to their potency as calmodulin antagonists (Donohoe et al., 2006). In addition, the antipsychotic drugs to varying degrees block serotonin receptors, so upregulation of ser-7 and/or ser-1 in response to chronic blockade by drug may contribute to the rebound response. However, cyclosporin A and calmidazolium, which do not inhibit serotonin binding, produce a similar rebound response without affecting serotonergic receptors.
Inhibition of calcineurin either directly (with cyclosporin A) or indirectly (via antagonism of calmodulin) may increase the phosphorylation of TPH-1, thus stabilizing interactions with 14-3-3 proteins (Ichimura et al., 1987), which would reduce TPH-1::GFP turnover in NSM neurons. Here, we show that trifluoperazine, an established antagonist of calmodulin-calcineurin activity, decreases the turnover of TPH-1::GFP in our system. These observations are consistent with the recent suggestion of Zhang et al. (2005) that post-translational mechanisms may affect TPH-1 levels in the ADF neurons of C. elegans.
At the cellular level, we documented a drug-induced increase in TPH-1::GFP expression and the number and extent of varicosities in serotonergic neurons. Varicosities are defined as significant bulges or thickenings in neuronal processes and represent sites of neurotransmitter release (Oleskevich and Descarries, 1990; Bunin and Wightman, 1999). In some cases, they correspond to synaptic specializations (Nonet, 1999). Therefore, it is not surprising that withdrawal from drug is accompanied by a dramatic recovery and even overshoot in the rate of pharyngeal pumping. The increase in varicosities correlates with an increase in the accumulation of TPH-1 secondary to a decrease in protein turnover. However, the drugs may also stimulate TPH-1 gene expression (Donohoe et al., 2008). Regardless, these data show that antipsychotic drugs produce dynamic remodeling of serotonergic neurons including sites of neurotransmitter release.
In this report, we show that the calcineurin inhibitor, cyclosporin A, increases TPH-1::GFP expression and produces a significant rebound response in C. elegans that is mediated by serotonin. Thus, this drug appears to potently stimulate serotonergic function. These findings may have clinical relevance because patients treated with cyclosporin A to suppress immunity and graft rejection often develop a spectrum of side effects that have been linked to excessive serotonergic activity (Azzadin et al., 1996; Wong et al., 2002).
The observation that antipsychotic drugs induce novel cellular and molecular changes in serotonergic neurons may also help to explain paradoxical clinical effects of these medications. Antipsychotics are generally viewed as dopamine and serotonin receptor antagonists, yet they are often used to treat conditions characterized by serotonin insufficiency, e.g., treatment-resistant depression (Nemeroff, 2005) and suicidal behavior (Montgomery et al., 1992; Meltzer et al., 2003). In addition, the maximum clinical response to antipsychotic medications typically takes several weeks or more to achieve (Lieberman et al., 1993; Conley et al., 1997), despite the fact that full receptor occupancy by drug is reached within days (Coppens et al., 1991). Furthermore, the molecular basis for rebound psychosis - the exaggerated expression of psychotic symptoms upon sudden discontinuation of drug (Chouinard and Jones, 1980; Meltzer et al., 1996) - remains a mystery. The rebound response of C. elegans to withdrawal from antipsychotic drugs was mediated via serotonin and was blocked with methiothepin, a serotonergic antagonist. Interestingly, rebound psychosis in schizophrenic patients has similarly been treated with a serotonin receptor antagonist, cyproheptadine (Meltzer et al., 1996). If the antipsychotic drugs produce an increase in serotonin in humans as they do in our system, this might explain their therapeutic efficacy in treatment-resistant depression and suicide prevention. Moreover, time-dependent adaptive changes in tryptophan hydroxylase expression and gradual remodeling of varicosities in response to the drugs may account for the delay in therapeutic onset in patients. Finally, it is intriguing that the major components of the rebound response to antipsychotic drugs in C. elegans, calcineurin and tryptophan hydroxylase, constitute genetic risk factors for schizophrenia (Gerber et al., 2003) and suicidal behavior (Bondy et al., 2006) in psychotic patients.
We thank L. Avery for providing the ser-7 and ser-1;ser-7 mutant strains for these studies, the Caenorhabditis Genetics Center for all other mutant strains, E. Osborn for technical assistance with the initial pilot studies, and M. Driscoll and J. Rand for comments on an early version of the manuscript. This work was supported by a grant from NIH/National Heart, Lung and Blood Institute (D.S.D. and E.J.A.).
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