The SERT Met172 Mouse: An Engineered Model to Elucidate the

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The SERT Met172 Mouse: An Engineered Model to Elucidate the Contributions of Serotonin Signaling to Cocaine Action Linda D. Simmler, and Randy D. Blakely ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00005 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 4, 2019

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ACS Chemical Neuroscience Simmler and Blakely, SERT Met172 model

The SERT Met172 Mouse: An Engineered Model to Elucidate the Contributions of Serotonin Signaling to Cocaine Action

Linda D. Simmler1 and Randy D. Blakely2*

Departments of 1Basic Neurosciences, University of Geneva, Rue Michel-Servet 1, 1206 Geneva, Switzerland and 2Biomedical Science, Charles E. Schmidt College of Medicine and Brain Institute, Florida Atlantic University, Jupiter, FL USA

*Corresponding author Randy D. Blakely, Ph.D. Rm 109, MC-17 5353 Parkside Dr. Jupiter, FL 33458 TEL: 561-799-8100 Email: [email protected]

Keywords: cocaine, serotonin, transporter, pharmacology, Slc6a4, knock-in, Met172

ACS Paragon Plus Environment

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Simmler and Blakely, SERT Met172 model

Abstract: Cocaine abuse and addiction remain highly prevalent and, unfortunately, poorly treated. It is well known that essential aspects of cocaine’s addictive actions involve the drug’s ability to block the presynaptic dopamine (DA) transporter (DAT), thereby elevating extracellular levels of DA in brain circuits that subserve reward, reinforcement, and habit. Less well appreciated are the multiple DA-independent actions of cocaine, activities that we and others believe contribute key pieces to the puzzle of cocaine addiction, treatment and relapse. In particular, a significant body of work points to altered serotonin (5-HT) signaling as one such component, not surprising given that, relative to DAT, cocaine acts as potently to block the 5-HT transporter (SERT) as to block DAT, and thereby elevates extracellular 5-HT levels throughout the brain when reward-eliciting DA elevations occur. To elucidate the contribution of SERT antagonism to the actions of cocaine, we engineered a mouse model that significantly reduces cocaine potency at SERT without disrupting the expression or function of SERT in vivo. In this short perspective, we review the rationale for development of the SERT Met172 model, the studies that document the pharmacological impact of the Ile172Met substitution in vitro and in vivo, and our findings with the model that demonstrate serotonergic contributions to the genetic, physiological and behavioral actions of cocaine.


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1. Introduction Cocaine is a popular drug of abuse and presynaptic dopamine (DA) transporter (DAT) (DAT) inhibitor that, acutely, induces euphoria and reinforcement, but where chronic use leads to addiction-associated adaptations in the structure and function of brain circuits.1 Although studies in animal models and in humans have amply demonstrated the ability of cocaine to elevate extracellular DA as a consequence of DAT blockade, cocaine, a prototypical ‘dirty drug’, interacts with other targets that studies show to be of consequence in understanding cocaine-induced behavioral changes.2-4 Given their close genetic relationship to DAT, it is not surprising that both the norepinephrine (NE) and serotonin (5-HT) transporters (NET and SERT, respectively) interact with cocaine, at much the same potency.5 These interactions are not inconsequential for understanding the physiological responses to cocaine. For example, sympathetic NET blockade and excessive NE elevations contributes to fatal arrhythmias and cardiomyopathies suffered by some cocaine abusers.6 NET blockade by cocaine not only attenuates NE clearance, but also likely impacts DA clearance in brain circuits where NET, and not DAT, transports DA.7-9 The contribution of SERT blockade to the actions and abuse liability of cocaine has been debated for decades,10,

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owing both to the non-reinforcing actions of SSRIs and the complexity of 5-HT

receptor signaling and its pharmacology. Indeed, prior work has often suggested that 5-HT effects oppose those of DA in relation to reinforcement,12 but given the diversity of 5-HT receptors and the complexity of serotonergic projections, dualistic models are clearly too simplistic. The contribution made by serotonergic mechanisms to the actions of drugs of abuse has become additionally relevant with the emergence of a large class of novel psychoactive substances (NPS) termed generically “bath salts” that have varying degrees of monoamine transporter selectivity, as well as differences in capacity for neurotransmitter uptake inhibition versus efflux induction.13, 14 Whereas cocaine exhibits relatively balanced potency at SERT and DAT,

the

cathinone-derivative

(‘legal

high’)

3,4-methylenedioxypyrovalerone

(MDPV)

demonstrates high selectivity for DAT over SERT, whereas other NPS such as mephedrone


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demonstrate a greater SERT selectivity, aligning in pharmacology with the amphetaminederivative 3,4-methylenedioxymethamphetamine (MDMA, ‘ecstasy’). A general hypothesis from the preclinical and clinical experience with these drugs predicts that selectivity for DAT over SERT suggests high abuse liability, whereas selectivity for SERT over DAT tracks with low abuse liability.15,

16

In the case of monoamine transporter inhibitors, this hypothesis is supported by

findings with rats that self-administered cocaine-derivates with different relative potencies at DAT versus SERT.17 The more selective a derivative was for DAT over SERT, the more readily it was self-administered, as quantified by progressive ratio schedule. The same was evident for analogs of amphetamine-derivatives and as such monoamine releasers, indicating again that actions at SERT can reduce the abuse liability of a compound.18,

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These studies support the idea that

nuanced approaches that target one or more 5-HT receptors could diminish compulsive drug seeking, addiction and/or relapse.20

2. Limitations of conventional approaches to assess SERT-mediated cocaine effects Different approaches have been used to assess the contribution of 5-HT signaling to cocaine action, including the complete genetic deletion of SERT in mice and rats.21-25 Lesion of the 5-HT neurons with 5-HT-specific toxins have also been utilized to diminish 5-HT contributions to drug action,

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as well as reduction of 5-HT levels by inhibition of 5-HT synthesis.27 To target 5-HT

signaling downstream of elevated extracellular 5-HT levels, selected 5-HT receptors are often antagonized.28 Several excellent reviews have summarized these lines of research and their findings.11, 29, 30 We can expect to see in future studies the optogenetic or chemogenetic (i.e., DREADD) inhibition of 5-HT neurons as a strategy to disable serotonergic contributions to cocaine action.31 Each of these approaches, however, come with disadvantages, including molecular and circuit level compensations that arise in the context of lifelong serotonergic alterations or with changes that alter broadly the signaling capacity of 5-HT systems. Any model that involves alterations of basal 5-HT neuron firing, secretion or clearance limits conclusions on the role of 5-


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HT in cocaine action to a context of hypo- or hyperserotonergic states. As a case in point, SERTKO mice show significant alterations on a cellular and behavioral level and differ in their basal behavioral phenotypes from wildtype littermates.32 Due to constitutive KO of SERT, baseline 5HT levels are elevated in basal (non-drug) condition and other transporters can take over for 5HT uptake.33 5-HT receptor manipulations, while well aligned to potential therapeutic strategies, bring with them the issue of disrupting just one out of many 5-HT signaling systems. Lesion and depletion strategies, and more sophisticated opto/chemogenetic paradigms to limit 5-HT neuron firing, may have broader effects than desired. Chronic cocaine reduces the basal activity of 5-HT neurons compared to the drug-naïve state,29 which has implication for drug-adaptive behavior. Therefore, manipulations of basal 5-HT signaling capacity, even for the purpose of assessing the role of 5-HT in cocaine action, may thus interfere with naturally occurring, cocaine-induced adaptations of 5-HT systems, obscuring meaningful effects that come on line with chronic exposure. Ideally, to circumvent the complexity of basal alterations in 5-HT signaling arising from gene knockout, lesion and drug manipulations, it would be ideal to achieve a “pharmacological knockout” strategy, where SERT could be modified to reduce cocaine sensitivity without impacting SERT function. Such strategies have been followed for several other drug targets, including DAT,34 nicotinic receptors,35 and GABAA receptors.36 For example, mice expressing a cocaineinsensitive DAT have provided strong support that DAT inhibition is required for cocaine-induced drug-adaptive

plasticity,37

preference (CPP).34,

39, 40

self-administration,38

hyperlocomotion

and

conditioned

place

Similarly, mice bearing a GABAA receptor mutation impacting

benzodiazepine interactions demonstrate that these agents exert their addictive potential via GABA neurons in the VTA, thereby indirectly activating VTA DA neurons.41 To pursue such studies with respect to cocaine/SERT interactions requires that a site for mutation be identified and a specific substitution engendered that can disable drug interactions, but not impact neurotransmitter binding and transport. Below, we describe our discovery, development, and use


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of the SERT Met172 mutation, which satisfies this requirement with respect to both antidepressants and cocaine.

3. Development and validation of the SERT Met172 model Shortly after rodent and human SERT cDNAs were cloned, and antidepressants and cocaine validated as SERT antagonists in heterologous expression systems,42, 43 our work with the Niznik lab led to the elucidation of the structure and pharmacology of a Drosophila melanogaster SERT ortholog.44 Conspicuously, while the apparent affinity of 5-HT for fly SERT was approximately the same as we had observed with mammalian SERTs, species differences in sensitivities to a number of antidepressant drugs and cocaine analogs were evident. Since antidepressants are competitive antagonists of 5-HT uptake, our findings suggested that residues that lie near the substrate binding site may have diverged to differentially impact antidepressant but not 5-HT binding. Through a series of “species-scanning” mutagenesis experiments, we identified two amino residues within transmembrane domains I and III (Y95 and Ile172, respectively), that conferred human SERT-specific, high-affinity recognition of antidepressants.45, 46

Mutagenesis to engineer fly amino acid identities at these sites (Phe95 and Met172,

respectively) in the human SERT cDNA dramatically reduced antidepressant potency while retaining normal 5-HT interactions.

Interestingly, we found that the human and mouse SERT

possessing the Ile172Met substitution not only displayed a loss of antidepressant sensitivity, but also showed a significant loss of cocaine potency (Figure 1).46 These and subsequent findings 47 were the first to provide structural data to indicate that transmembrane domains I and III contributed to the binding pockets for substrates and structurally distinct competitive antagonists, conclusions later validated with the elucidation of monoamine transporter crystal structures.48-50 Moreover, they suggested the possibility of eliminating antagonist binding at SERT in vivo without disabling normal SERT function, and thereby allow for tests of the role of SERT and 5-HT signaling in both antidepressant and cocaine action.


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Our next goal was to obviously bring this hypothesis to a test, for which we appropriated mouse embryonic stem cell-based homologous recombination approaches to knock in sequences that encode Met172 in the endogenous mouse SERT (Slc6a4) locus.51 We generated homozygous Met172 SERT mice (and wildtype (Ile172) littermates) and in our initial report, demonstrated, as expected, no effect of mutation on SERT protein expression, 5-HT uptake kinetics or tissue 5-HT levels. When the sensitivity of SERT Met172 synaptosomal 5-HT uptake to antagonists was queried, however, significant reductions in potency for demonstrated for the antidepressants fluoxetine and citalopram, as well as for cocaine, compared to wildtype preparations, matching expectations from transfected cell studies. To bolster our interpretations that the SERT Met172 mutation mice disabled antidepressant interactions with the transporter in vivo, we pursued in vivo microdialysis studies.51 In wildtype mice, systemic administration of fluoxetine or citalopram led to a robust elevation of extracellular 5-HT. These effects were completely lost in the SERT Met172 mice. Inactivity of blockers at SERT Met172 was further validated in vivo using chronoamperometry 51, 52 and ex vivo with slice electrophysiology 51. Using the forced-swim test and tail suspension test, assays that reveal the actions of acutely administered antidepressants, we demonstrated a loss of drug (citalopram, fluoxetine) action in the SERT Met172 model.51 Using the novelty induced hypophagia test and hippocampal stem cell proliferation assays, tests sensitive to chronic antidepressant administration, we again observed a loss of antidepressant activity in the SERT Met172 mice.53 These findings support an essential role of SERT targeting for the antidepressant actions of these drugs. As with in vitro studies,46 the mutation did not compromise the behavioral actions of the antidepressant paroxetine, providing a useful, active control in these and future studies. Recently, the SERT Met172 model has demonstrated value in showing that vortioxetine, a novel antidepressant with direct effects on 5-HT receptors (in addition to SERT inhibition), does not depend on SERT antagonism for antidepressant-like effects.52 Lastly, the SERT Met172 model also proved


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valuable in demonstrating that the ability of citalopram to modulate axon guidance is SERTindependent, mediated instead by the drug’s interactions with a sigma receptor.54

5. Use of SERT Met172 mice to implicate 5-HT signaling in cocaine actions Given the utility of SERT Met172 mice in validating a role for SERT in antidepressant action, we turned our attention to the potential use of the model in demonstrating SERT contributions to cocaine action. In a first study, the SERT Met172 model was used to demonstrate an essential role of SERT in the ability of cocaine to shift circadian rhythm. Earlier work with 5-HT receptor antagonists and in vitro systems suggested that cocaine dysregulates the circadian clock via 5-HT signaling.55 With the SERT Met172 model, which is devoid of non-specific effects that can be a confounder when 5-HT receptor antagonists are used, Prosser and colleagues gathered evidence using hypothalamic brain slice electrophysiology and wheel-running behavior that cocaine binding to SERT is essential for the cocaine-induced changes in rhythms evident in these preparations.56 Subsequently, we pursued in vivo microdialysis studies and, as was observed with antidepressants, i.p. administration of cocaine proved unable to elevate extracellular 5-HT levels in the SERT Met172 mice at a concentration that produced a robust elevation in wildtype animals (Figure 2A).57 With the confirmation that cocaine does not induce elevation of 5-HT levels in SERT Met172 mice in vivo, we assessed whether SERT inhibition plays a role in behavioral alterations that are typically observed in mice after cocaine administration. Acute cocaine-induced hyperlocomotion and behavioral sensitization to cocaine did not differ between SERT Met172 and wildtype mice, consistent with these behavioral responses arising foremost from DAT inhibition. In contrast, CPP, achieved after 4 days of conditioning with cocaine interleaved with 4 days of opposite chamber conditioning with saline, was more pronounced in SERT Met172 mice (Figure 2B) than in wildtype littermates. These findings indicate that important SERT (and therefore 5-HT signaling) engagement normally contributes a negative influence on context associations with


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cocaine and support the possibility that the strength of associations made by humans to the context of drug administration may be influenced by genetic or environmental influences on 5-HT systems. Extinction of cocaine place preference after 8 days of conditioning with saline occurred to the same extent in SERT Met172 and wildtype littermates, but reinstatement with an acute cocaine injection restored stronger CPP in SERT Met172 mice, compared to wildtype. We made a similar observation in a 2-bottle choice paradigm, in which a different cohort of animals was given ad libitum access to a bottle with water only and a bottle containing dissolved cocaine. In the second week of the choice-test, SERT Met172 mice voluntarily consumed more cocainesupplemented water than the wildtype littermates, which could be interpreted as stronger reinforcing effects of cocaine with absent SERT inhibition. Possibly related to this behavioral response, we observed that in some brain areas, including the prelimbic cortex (Figure 2C and D), piriform cortex and the dorsal hippocampal CA3 region, SERT Met172 mice exhibited higher cocaine-induced c-Fos expression than observed with wildtype control littermates. C-Fos is a marker for neuronal activation and this finding indicates, similar to the CPP test, SERT blockade by cocaine depresses effects that arise from other cocaine targets, such as from DAT (or NET) inhibition (Figure 2E). We also observed that thigmotaxic locomotion after cocaine administration is higher in SERT Met172 mice, compared to littermate controls. These data suggest that cocaine normally exerts a SERT-dependent anxiolytic effect that is lost in the cocaine-treated SERT Met172 mice. To capture a broader pattern of circuit-specific molecular changes that arise after cocaine administration and that are SERT-dependent, we performed transcriptome-wide RNASeq analyses along with candidate gene mRNA expression studies,57 focusing on the prefrontal cortex and ventral striatum. These studies revealed cocaine-elicited, SERT-dependent, transcriptional programs that were both region and time-dependent. Space does not permit a detailed discussion of the nature of the gene expression networks that were identified as dependent on SERT antagonism, but several interesting patterns emerged. A role for SERT inhibition by cocaine was


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evident for networks linked to MAP kinase (Erk1/2, Jnk) and Akt with both acute and chronic cocaine administration. These kinases have been associated in prior studies with cocaineinduced signaling and morphological plasticity, indicating that further evaluation of their serotonergic control is warranted and may lead to novel targets for medication development. We also identified a NFB linked network as one through which SERT antagonism by cocaine changes gene expression. This finding is interesting given that NFB is activated by FosB, a gene product implicated in cocaine-induced neuronal plasticity. While these networks have been previously linked to cocaine reward and addiction, their emergence as specifically linked to the serotonergic influences of cocaine suggests that 5-HT actions are integral to the molecular program engaged to reach and maintain an addicted state. Our behavioral findings following cocaine administration using the SERT Met172 model are overall in line with studies using SERT-KO animals.11 Despite the compensatory alterations in 5-HT signaling that arise in the SERT-KO,32 studies with these mice suggested that SERT inhibition is not required for cocaine reinforcement, as measured through cocaine selfadministration,58 and hyperlocomotion, 24 but that SERT inhibition in WT animals normally reduces CPP.23, 24 We reached the same conclusion with respect to cocaine hyperlocomotion and CPP using the SERT Met172 mice, a model where cocaine interactions with SERT are precluded in the absence of tonic elevations in extracellular 5-HT. Together, these findings argue that, although many changes occur in the brain of animals deprived of SERT throughout life, these changes are not ones required for SERT inhibition to limit cocaine CPP. Moreover, these findings indicate that direct comparisons between the SERT-KO and the SERT Met172 can be useful in understanding whether lifelong changes in SERT-mediated 5-HT clearance, versus direct SERT antagonism, underlines drug action. Our studies examining the impact of SERT Met172 on the circadian actions of cocaine and the ability of SERT to modify CPP illustrate two distinct uses of the knock-in model. In the


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former study, SERT Met172 allows for a validation that SERT (versus DAT, NET or other targets) is essential for cocaine actions.56 In the latter study, the knock-in model demonstrates that SERT contributes to a behavioural outcome that is primarily driven by cocaine action elsewhere (on DAT primarily).57 Another example of the latter, modulatory contribution of SERT to cocaine-elicited behavior, and one that arises as an effect of disease-associated perturbations in DA signaling, was recently reported by Stewart and colleagues who appropriated the SERT Met172 model to demonstrate that an emergent cocaine-induced suppression of locomotor activation seen in the DAT Val559 mouse arises due to cocaine action at SERT.59 The DAT Val559 variant has been reported in individuals with bipolar disorder,60 ADHD,61 and autism spectrum disorder 62 and found to confer anomalous DA efflux.63 Mergy and colleagues,64 using DAT Val559 knock-in mouse, demonstrated that the mutation elevates basal extracellular DA levels in the striatum in vivo, and produces a number of physiological and behavioral alterations, as well as a blunted locomotor responses to D-amphetamine and methylphenidate. Stewart and colleagues

59

found that a

locomotor stimulation was completely absent in the DAT Val559 mice, and reasoned that the difference between the blunted locomotor actions of the DAT antagonist methylphenidate and the absent locomotor stimulation produced by cocaine could be due to the additional SERT actions of cocaine, a surmise validated when cocaine injections given to DAT Val559/SERT Met172 double mutants restored locomotor activity.

6. Role of SERT antagonism in cocaine abuse – next steps with the SERT Met172 model To date, using the SERT Met172 model, the tests of SERT-dependence in relation to cocaine-induced behavioral changes have utilized non-contingent drug administration protocols, assessing locomotor activation patterns, locomotor sensitization, and context-association with drug delivery.56,

57

Although these tests provide information on the ability of 5-HT signaling to

contribute to some mechanisms of cocaine actions, cocaine self-administration is the “goldstandard” paradigm to measure reinforcement and the addictive potential of drugs of abuse. Use


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of the SERT Met172 mice in such a paradigm, as continuation of the study conducted by Thomsen et al. using SERT-KO mice,58 will allow for a more direct and translationally relevant contribution of serotonergic changes to the acquisition of self-administration (acute reinforcement), the dose self-administered (dose titration; sensitivity of SERT Met172 mice to cocaine), in progressive ratio testing (motivation to self-administer) and in cue-induced seeking after withdrawal (drug-adaptive plasticity). A key facet of substance abuse disorder is the pursuit and use of drugs despite the knowledge of negative consequences.65 By introducing punishment to the self-administration paradigm, for example by delivering a foot shock with the cocaine infusion, compulsive drug seeking and use despite knowledge of negative consequences can be assessed. If the hypothesis of a protective effect of SERT inhibition regarding the abuse liability of a drug holds true, we predict that a greater proportion SERT Met172 mice will become compulsive drug seekers, compared to wildtype mice, effects that can then lead to studies of specific serotonergic circuits and receptors involved. Typically, only about 20% of rodents self-administering cocaine continue after introduction of punishment,66, 67 which reflects also the population of compulsive cocaine consumers in humans. In contrast, self-stimulation of DA neurons, a model for addiction, produces a higher population (60-70%) of compulsive mice.67, 68 It is possible that, in addition to differences in kinetics, the action of cocaine at SERT is responsible for a lower percentage of compulsive animals, compared to the DA self-stimulation model which is independent of any 5-HT effects. Our finding on higher CPP scores in SERT Met172 animals

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predict stronger reinforcement of

cocaine as assessed in the self-administration test. Interestingly, a cocaine effect selectively mediated by 5-HT has recently been proposed based on slice in vitro experiments. Matsui and Alvarez showed that acute cocaine induces 5HT1B receptor -mediated, presynaptic depression of the nucleus accumbens-to-ventral pallidum projection in D2 receptor-expressing MSNs (D2-MSNs).69 This effect is 5-HT dependent, as pure DAT and NET inhibitors did not induce the presynaptic depression, but the SSRI citalopram as well as endo- and exogenous 5-HT did. Further experiments are required to assess the behavioral


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implication and link to long-term drug-adaptive effects. The SERT Met172 mouse model could be a useful model to investigate these 5-HT mediated effects in vivo. As to translational value, the observations by Matsui and Alvarez may relate to cocaine-induced anhedonia, which relies on depression of the D2-MSNs nucleus accumbens -to-ventral pallidum projection.70 Relapse is a critical issue for the treatment of drug abuse, as, even after years of abstinence, drug users are prone to return to their drug taking habits. The 5-HT system has been implicated in relapse vulnerability in two ways.20 First, by playing a role in impulsivity, cortical 5HT2A and 5-HT2C receptors affect inherent vulnerability to relapse.71, 72 Second, cocaine-evoked changed on 5-HT2C receptor function appear to contribute to drug relapse.73 Due to the complexity of 5-HT receptors and the opposing effects of 5-HT2A and 5-HT2C receptors in impulsivity,74 the SERT Met172 model may be an ideal tool to investigate whether SERT inhibition by cocaine plays a role in relapse vulnerability. Both a protective and a permissive role of SERT inhibition is conceivable, which might also depend on the status of inherent vulnerability of the animals (and humans) due to individual expression levels of 5-HT receptor subtypes. To relate SERT-mediated cocaine effects to relapse vulnerability, cocaine-treated SERT Met172 mice could be tested for emergence (or not) of greater cue reactivity, a predictor for relapse, and if so, whether specific 5HT receptor actions that support relapse are involved. The SERT Met172 model seems the least fraught with compensatory issues to conclusively answer whether relapse to cocaine use relies on a serotonergic target, and can add weight to efforts to develop serotonergic pharmacotherapies to support abstinence.75

7. Conclusion The SERT Met172 mouse model has been useful for the dissection of SERT-mediated effects of antidepressants and cocaine.52,

53, 56, 57

While the studies noted are obviously from

animal models, we have attempted to stress their translational value. For the novel antidepressant vortioxetine which acts at 5-HT receptors and at SERT, the SERT Met172 model showed that


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SERT inhibition is not required for the molecule’s antidepressant effect. One inference of the latter study is that SERT inhibition might be eliminated from vortioxetine derivatives for the development of next generation antidepressants, possibly further improving side-effect profile and wider utility. At minimum, use of the SERT Met172 model can provide strong, supportive data that SERT antagonism is or is not critical to the actions of any mixed action antidepressants developed, leading to further medicinal chemistry efforts to retain or remove SERT targeting as drug development proceeds. The dissection of SERT-mediated effects in cocaine action, including drug seeking and compulsive drug use, could also point towards novel directions to treat cocaine abuse. Should the hypothesis of a protective effect of 5-HT signaling through specific receptor subtypes in drug abuse hold true, self-administration in the presence of negative reinforcers, a rationale to generate 5-HT receptor-targeted therapeutic drugs would be bolstered. A comparative use of neuronal activation markers such as c-fos and Arc across the full CNS may help identify specific brain regions where SERT antagonism contributes to cocaine action, helping to identify SERT-dependent, cocaine-modulated circuits that can receive further attention in basic rodent and human studies, and ultimately identify novel sites for targeted therapies such as deep brain stimulation or virally-mediated chemogenetic strategies. Finally, transcriptional networks differentially activated or suppressed by the SERT Met172 mutation in future self-administration paradigms may identify other druggable targets for medication development that might escape detection otherwise.

Conflict of interest LDS and RDB declare no competing financial interest. RDB served on the Psychopharmacology Advisory Board of Lundbeck during the development of vortioxetine, a drug noted in the paper. He no longer serves in that capacity nor receives any ongoing monetary support for this activity.

Acknowledgements


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LDS is supported by the Swiss National Science Foundation (PZ00P3_174178). RDB is supported by NIH Awards MH094527 and MH105094.

Author contributions LDS and RDB wrote the manuscript.

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Figure 1: Amino-acid substitution in the transmembrane III (TM3) of SERT that hinders high-affinity recognition of cocaine. A: Alignment of amino acids (top panel) that are part of the cocaine binding pocket in human and mouse SERT (bottom left). Ile is expressed at the 172 position. In synaptosomal preparation from wildtype mice, cocaine inhibits 5-HT uptake with a potency in the nanomolar range (bottom right). B: Alignment of amino acids (top panel) that are part of the cocaine binding pocket in mouse SERT with the Ile172Met substitution and in drosophila SERT. Met is expressed in the mutated mouse SERT and in SERT from drosophila. The bulky amino acid Met sterically hinders high-affinity binding of cocaine, as illustrated in the model of the binding pocket (bottom left). In synaptosomal preparation from SERT Met172 mice (bottom right), cocaine inhibits 5-HT uptake with a potency in the micromolar range, being 80-fold less potent than in wildtype. This figure is adapted from Henry et al. 46 (Copyright 2006 American Society for Biochemistry and Molecular Biology) and Simmler et al. 57 (Copyright 2017 John Wiley and Sons).

Figure 2: Key findings from cocaine effects in SERT Met172 mice. Figures are adapted from Simmler et al.

57

(Copyright 2017 John Wiley and Sons). A: Applying microdialysis in the

hippocampus, elevated extracellular 5-HT levels are evident after intraperitoneal administration of cocaine in wildtype (SERT Ile172) animals. In contrast, cocaine does not change 5-HT levels in SERT Met172 animals due to the ‘cocaine-insensitivity’ of the mutated SERT. B: Cocaineinduced hyperlocomotion and behavioral sensitization did not differ between wildtype and SERT Met172 animals. C: In a classic conditioned place preference test, SERT Met172 mice showed higher preference for the cocaine-paired chamber than wildtype littermates. Data is expressed as difference in time spent in the cocaine-paired side after vs. before conditioning, during a 20 min test session. D: Representative pictures of brain slices (prelimbic cortex) immunostained for cFos 120 min after injection of saline or cocaine. E: Quantification of c-Fos positive cells in the prelimbic cortex after saline or cocaine treatment in wildtype and SERT Met172 mice. F: Simplified


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schematic illustrating the putative role of SERT inhibition for neuronal activity in the prelimbic cortex and in conditioned place preference. In wildtype animals (left), elevated 5-HT levels after cocaine partially suppress cocaine-induced effects arising from elevated DA levels. In cocainetreated SERT Met172 mice (right), 5-HT levels remain low and effects arising from high DA (or NE) levels are unopposed and therefore stronger than in wildtype animals. * P < 0.05, significantly different from day 1 (panel B), wildtype (panels C and E), or from saline control (panel E), as indicated; Sidak post hoc test following 2-way ANOVA.


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ACS Chemical Neuroscience

For Table of Contents Use Only

SERT Ile172 (WT SERT)

DAT

NET

SERT

cocaine

cocaine

cocaine

Cocaine

SERT Met172 (SERT-KI)

DAT

NET

cocaine

cocaine

SERT Met172

‘Pharmacological KO’; cocaine-insensitive SERT Cocaine



Figure 1

TM3 human 157 mouse WT 157

TM1

I F K G I G Y A I C I I A F Y I A S Y Y N T I M AWA L Y Y L I F K G I G Y A I C I I A F Y I A S Y Y N T I I AWA L Y Y L

COC

SERT Ile172 (WT)

100

TM6

TM8 I172

% 5-HT uptake

A

TM10

50

0 TM3

-9

B

-8

-7 -6 -5 Cocaine [M]

-4

-3

TM3

mouse Met172 157 I F K G I G Y A I C I I A F Y M A S Y Y N T I I A W A L Y Y L drosophila 111 A L K G V G Y A I C L I D I Y M G M Y Y N T I I G W A V Y Y L

TM1

COC

TM8 M172

SERT Met172 (KI)

100

TM6

% 5-HT uptake

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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50

TM10 TM3

0 -9

-8

-7 -6 -5 Cocaine [M]


-4

-3

Page 27 of 27

Figure 2

300 200 100 -40 -20 0

D

Prelimbic cortex (PL) WT saline

Met172 saline

F

20 40 60 80 100 120 140 Time (min)

WT cocaine

Met172 cocaine

Cocaine-treated WT mice

5-HT

DA

Neuronal activity in PL Conditioned place preference

100

*

time in cocaine-paired chamber (s)

400

C

SERT Ile172 (WT) SERT Met172 (KI) *

75 50 25

E

SERT Ile172 (WT) SERT Met172 (KI)

600

400

200

0

c-Fos stained cells per square pixel in PL

Cocaine i.p.

125

0

se lin D e ay D 1 C ay ha 5 lle ng e

500

B


Total distance traveled (m)

600

Ba

A Dialysed 5-HT (% baseline)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Post conditioning

*

0.004

* *

0.003


0.002 0.001 0.000

Saline

Cocaine

Cocaine-treated SERT Met172 mice

5-HT

DA

Neuronal activity in PL Conditioned place preference


The SERT Met172 Mouse: An Engineered Model to Elucidate the

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