Elsevier

Neuropharmacology

Volume 168, 15 May 2020, 107998
Neuropharmacology

Antidepressant-like effects of ketamine in a mouse model of serotonergic dysfunction

https://doi.org/10.1016/j.neuropharm.2020.107998Get rights and content

Highlights

  • 5-HTT knockout (KO) mice had increased immobility on the forced-swim test (FST).

  • Sertraline did not produce antidepressant-like effects in 5-HTT KO mice.

  • Ketamine had antidepressant-like effects in both wild-type and 5-HTT KO mice.

  • 5-HTT KO mice showed a decreased locomotor response to acute ketamine and MK-801.

  • Hippocampal GluN2A protein levels were reduced in 5-HTT KO mice.

Abstract

Traditional monoaminergic treatments of depression frequently exhibit suboptimal tolerability and effectiveness. The ‘short’ (s) allele variant of 5-HTTLPR is known to compromise transcriptional efficacy of the serotonin transporter (5-HTT) and can reduce treatment response to traditional antidepressants (e.g. selective serotonin reuptake inhibitors or SSRIs). This study sought to establish the 5-HTT knock-out (KO) line as a mouse model of SSRI-resistant depression and assess its response to a novel glutamatergic antidepressant, ketamine, a non-competitive N-methyl-d-aspartate receptor (NMDAR) antagonist. Following acute antidepressant treatment, 5-HTT KO mice and wild-type (WT) controls were subjected to the forced-swim test (FST), one of the most widely used techniques to detect acute antidepressant response. As hypothesised, when assessed 30 min after administration in the FST, the SSRI sertraline (20 mg/kg, i.p.) produced antidepressant-like effects in WT control but not in 5-HTT KO mice. In contrast, ketamine (20 mg/kg, i.p.) induced antidepressant-like effects in both genotypes. 5-HTT KO mice also exhibited a reduced locomotor response to both MK-801 (another NMDAR antagonist) and ketamine, and reduced GluN2A protein levels in the hippocampus, suggesting glutamatergic dysfunction in this model. These results highlight the utility of 5-HTT KO mice as a relevant model of SSRI-resistant depression and demonstrate that ketamine can produce acute antidepressant-like effects in conditions of 5-HTT deficiency. These findings extend existing literature that indicates ketamine is effective in ameliorating symptoms of treatment-resistant depression and may have implications for understanding the cellular and molecular mechanisms underlying the antidepressant effects of ketamine.

This article is part of the special issue entitled ‘Serotonin Research: Crossing Scales and Boundaries’.

Introduction

The recent development of rapid-acting antidepressants holds the promise of being a defining breakthrough in psychiatric treatment and management. While it is clear that major depressive disorder (MDD) is a uniquely pervasive and devastating condition (World Health Organization, 2017), there regrettably is a dearth of readily available treatments that demonstrate reliable efficacy. Indeed, current therapies achieve remission in only approximately two thirds of patients (Rush et al., 2006), with latency often in the order of weeks. Thus, treatment-resistant depression (TRD) has become the focus of novel therapeutic approaches.

The serotonin transporter (5-HTT) is the target of many traditional monoamine antidepressants, including selective serotonin reuptake inhibitors (SSRIs) (Schafer, 1999). 5-HTT regulates the serotonergic system, and modifications to functional 5-HTT expression are implicated in psychiatric illness (Kenna et al., 2012). The human SLC6A4 gene, which codes for the 5-HTT protein, contains a polymorphic region known as 5-HTTLPR, which may modify 5-HTT transcriptional efficacy and 5-HTT mRNA and protein expression (Lesch et al., 1996). The lower transcription ‘short’ (‘s’) allele is often associated with an increased vulnerability to mental illness and trait anxiety (Schinka et al., 2004). Some authors have reported an increased frequency of depression among carriers of the s allele (Lotrich and Pollock, 2004), however this effect appears subtle, and may emerge only in the presence of environmental stressors (Caspi et al., 2003; Nguyen et al., 2015). Of note, while a recent genome-wide association study (GWAS) of MDD did identify several relevant polymorphisms related to the targets of antidepressant medications, 5-HTTLPR was not among 44 genetic risk variants of the disease (Wray et al., 2018). It may be instead that the greatest clinical utility of 5-HTTLPR is through its ability to predict antidepressant treatment response, with s allele carriers shown to respond more poorly to SSRIs (Serretti et al., 2007), suggesting the polymorphism's candidature as a risk-factor in treatment-resistant presentations of depression.

The consequences of reduced 5-HTT expression have been investigated experimentally through the use of 5-HTT knockout (KO) mice, which lack 5-HTT mRNA and protein (Kästner et al., 2015). This mouse model exhibits a greater than 50% reduction in serotonin (5-HT) concentrations across most brain regions (Kim et al., 2005; Renoir et al., 2008), along with altered 5-HT receptor function (Mathews et al., 2004). 5-HTT KO mice display anxiety- and depressive-like behaviours (Carroll et al., 2007; Lira et al., 2003; Renoir et al., 2008; Rogers et al., 2017), although this phenotype may vary depending on genetic background (Holmes et al., 2002b). As expected, 5-HTT KO mice have been shown to exhibit a decreased response to the SSRI fluoxetine on the tail-suspension test, while the effects of tricyclic antidepressants desipramine and imipramine appear to be sustained (Holmes et al., 2002b). These mice also show reduced cortical and subcortical binding of SSRIs such as citalopram (Renoir et al., 2008). As such, 5-HTT KO mice provide a model of relevance to clinical populations who do not respond to SSRIs, which are currently recommended as the first-line pharmacological treatment for MDD (Cipriani et al., 2010).

Limitations of current treatments for depression have broadened aetiological discussion beyond that of the well-researched monoamine systems. There is growing support for the notion that altered synaptic plasticity and cellular resilience may play a causal role in depression (Manji et al., 2000), implicating the salience of neurotrophins such as brain-derived neurotrophic factor (BDNF). Glutamatergic markers have also been extensively studied, with emerging evidence of disrupted glutamatergic signalling and changes in N-methyl-d-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression and function in depression (Sanacora et al., 2008).

Further evidence of glutamatergic dysfunction in depression is yielded from the antidepressant effects of certain glutamatergic drugs. Ketamine is a non-competitive NMDAR antagonist that binds primarily to phencyclidine (PCP) sites near the channel pore (Kirkup and Bennett, 2012); it has long been used to induce anaesthesia, an effect linked primarily to its strong antagonism of the NMDAR (Yamamura et al., 1990). More recently, low doses of ketamine have been shown to rapidly reduce symptoms of depression in preclinical (animal model) studies and clinical trials (Iadarola et al., 2015; Machado-Vieira et al., 2009). Notably, glutamatergic signalling and novel antidepressants such as ketamine are yet to be assessed in the 5-HTT KO mouse model.

While the potential for ketamine to ameliorate symptomology in TRD (Mathew et al., 2012; Murrough et al., 2013) represents a significant breakthrough, its psychotomimetic/dissociative side-effects raise significant abuse liability concerns, particularly at high doses (Browne and Lucki, 2013). Interestingly, however, time-course (Berman et al., 2000) and active placebo (Murrough et al., 2013) studies have suggested a dissociation between psychotomimetic side-effects and the antidepressant response. Exploitation of the mechanism of ketamine's antidepressant properties in isolation may stimulate research into new glutamatergic drugs that do not exhibit ketamine's abuse profile.

Although not yet precisely defined, the mechanism of ketamine's antidepressant effect is likely to be, at least in part, induced by an AMPAR-dependent activation of mammalian target of rapamycin (mTOR) signalling and rapid BDNF release (with associated tropomyosin related kinase (TrkB) activation) (Abdallah et al., 2016; Duman et al., 2019; Lepack et al., 2014). The promotion of BDNF-induced plasticity is not unique to ketamine, and is in fact a sequela shared by many antidepressant treatments, including SSRIs (Duman et al., 2016).

Many studies have explored whether ketamine's antidepressant action is contingent on functional serotonergic transmission. While some have suggested serotonin-independent mechanisms (e.g. the 5-HT-independent effects of (R)-ketamine described in Zhang et al. (2017)), there appears to be a growing consensus of serotonergic involvement. Gigliucci et al. (2013) revealed that ketamine's ability to attenuate immobility on the forced-swim test (FST) was abolished in rats pre-treated with para-chlorophenylalanine (PCPA), a tryptophan hydroxylase inhibitor. In another important study, S. Yamamoto et al. (2013) demonstrated, using microdialysis, that a subanaesthetic dose of ketamine increased 5-HT transmission in the prefrontal cortex (PFC) in awake monkeys. This effect of ketamine to increase serotonergic transmission has been well-established in rodent models (Pham et al., 2017) and is likely to be mediated by AMPA receptors in the dorsal raphe nucleus (DRN) (Nishitani et al., 2014). A recent comprehensive review highlighted that ketamine initiates a complex series of interactions through the medial PFC-DRN network via AMPA, NMDA and gamma-aminobutyric acid (GABA) receptors to modify usual 5-HT transmission (Pham and Gardier, 2019). There is also a substantial body of literature documenting the role of various 5-HT receptor subtypes in the rapid antidepressant effects of ketamine that is beyond the scope of the present discussion. The link between the behavioural effects of ketamine and 5-HTT function remains unresolved; however, at anaesthetic doses ketamine has been shown to reduce reuptake of 5-HT via 5-HTT in rodents (Martin et al., 1990).

Taken together, these findings underscore the importance of investigating the efficacy of novel glutamatergic antidepressants in a model of monoamine dysfunction. The present study sought to establish the 5-HTT KO as a mouse model of SSRI-resistant depression, and also to determine whether ketamine's antidepressant-like effects would be sustained in 5-HTT KO mice. Although, as described above, there is a compelling account for the interaction of the serotonergic and glutamatergic systems in the context of depression, the currently understood model of ketamine's mechanism of action does not preclude effectiveness in conditions of serotonergic deficiency. We also note that while previous investigations such as Gigliucci et al. (2013) have investigated putatively transient changes to the serotonergic system, the use of the 5-HTT KO model enables investigation of the effects of dysregulated 5-HT during neurodevelopment, providing a unique perspective. We hypothesised that the antidepressant-like effects of ketamine would be evident in both wild-type (WT) and 5-HTT KO mice, the latter of which were predicted not to respond to SSRI treatment.

Secondarily, glutamatergic function (in particular, NMDAR activity) in 5-HTT KO mice was examined by assessing locomotor hyperactivity following acute ketamine and MK-801 (a non-competitive NMDAR antagonist) challenges. Ketamine-induced hyperlocomotion has relevance to the known psychotomimetic side-effects of the drug, while MK-801 provided additional evidence of NMDAR-specific function. In addition, hippocampal protein levels of important NMDAR subunits (GluN1, GluN2A and GluN2B) was assessed. Although not previously investigated in this particular mouse model, the known role of glutamate in the pathophysiology of depression led to the hypothesis that glutamatergic signalling may also be dysregulated in 5-HTT mutant animals. Should ketamine's antidepressant-like effect be evident in mice which exhibit both 5-HTT and NMDAR dysfunction, this may have implications for understanding the psychopharmacological mechanism of ketamine's antidepressant properties.

Section snippets

Animals

Male and female 5-HTT KO and WT control mice were bred on a C57BL/6J genetic background from a colony previously established at the Florey Institute of Neuroscience and Mental Health. After weaning, mice were housed in individually ventilated cages or open-top boxes in groups of two to five, according to sex. Behavioural testing commenced as mice reached maturity at approximately 8 weeks of age. Rooms were kept on a 12-h light/dark cycle (lights on at 7:00 a.m.) and access to food and water was

Sertraline

The antidepressant-like effects of sertraline were assessed using the FST. Unexpectedly, mice randomly allocated to the sertraline treatment group appeared to exhibit greater immobility at pretest (i.e. the day before drug administration), a trend which almost reached significance (F(1, 39) = 4.04, p = .051). To abate this, the progression of immobility behaviour across all three trials was assessed: 24 h prior to drug administration (pretest), 30 min post-administration and 24 h

Discussion

This study is the first to investigate ketamine efficacy and glutamatergic function in 5-HTT KO mice. As predicted, treatment with sertraline produced no change in FST performance in mice lacking the serotonin transporter (providing evidence to support an ‘SSRI-resistant’ model), while ketamine elicited an antidepressant-like effect in both WT and 5-HTT KO mice. Furthermore, 5-HTT KO animals were found to have reduced hippocampal GluN2A expression, and upon acute challenge exhibited a decreased

Declaration of competing interest

The authors have no conflict of interest to report.

Acknowledgements

This work was supported by an ARC Discovery Early Career Research Award (TR). AJH is an NHMRC Principal Research Fellow. The Florey Institute of Neuroscience and Mental Health acknowledges the support from the Victorian Government's Operational Infrastructure Support Grant.

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