Fast antidepressant action of ketamine in mouse models requires normal VGLUT1 levels from prefrontal cortex neurons
Graphical abstract
Introduction
Major depression is a mental disorder characterized by extreme low mood and anhedonia, which often occur together with a variety of other psychological and/or physiological disturbances. In recent years, major depression has become one of the leading causes of disability worldwide. Indeed, it has been estimated that 5% of the world's population suffer from this disease, while one-fifth experience at least one depressive episode throughout their life (Kessler and Bromet, 2013). The monoaminergic hypothesis of depression that links low monoamine levels, mainly 5-HT and NA, to depression has been the leading thesis for >50 years (Schildkraut, 1965). Yet, currently available monoaminergic antidepressants remain inadequate treatment for many patients. Only one-third of patients respond to the antidepressant treatment, showing an improvement >50% in the first 3–4 weeks and a complete remission of symptoms in a few months. Although another third of the patients do not respond well to the first-choice treatment, they do so when a second is administered or following combined therapy. Also, the last third of the patients necessitate multiple pharmacological strategies, as many of these suffer from treatment-resistant depression (TRD) (Rush et al., 2020) from the STAR*D Study Team. Consequently, it is essential to address TRD as an urgent social and medical need not only since it involves high levels of suffering in patients, their family members and friends, but also because it generates important socio-economic expenses worldwide.
The discovery of NMDA antagonists as rapid-acting antidepressant medications has revolutionized the approach to major depression (Berman et al., 2000; Lapidus et al., 2014; Loo et al., 2016; Murrough et al., 2013; Singh et al., 2016; Su et al., 2017; Zarate et al., 2006). Notably, on March 5, 2019, the FDA approved intranasal esketamine (Spravato™), the active enantiomer of ketamine, for adult TRD patients. In the same year, December 18, this medicine was also approved by EMA. However, not all TRD patients respond to esketamine and this drug requires close monitoring when administered due to its side effects that include dissociation, psychotomimetic properties and abuse potential (Sanacora et al., 2017).
In recent years, depression has been associated with deficient glutamate transmission in the PFC (Burgdorf et al., 2013; Koike et al., 2011; Maeng et al., 2008; Wolak et al., 2013; Zanos et al., 2016; Zanos and Gould, 2018). Mainly involved in decision-making and executive functions, the PFC is also an essential component of the neural circuitry modulating stress responses and emotions. Several pre-clinical studies in rodents have shown that ketamine has the ability to enhance glutamate transmission (Chowdhury et al., 2017; Moghaddam et al., 1997; Verma and Moghaddam, 1996) and trigger antidepressant-like effects within a few hours. De novo synthesis of the brain-derived neurotrophic factor (BDNF) might be involved in this antidepressant activity (Björkholm and Monteggia, 2016; Kim et al., 2021; Suzuki and Monteggia, 2020). However, the majority of pre-clinical studies looking at the molecular effects of ketamine have been carried out on healthy mice or in anxiety/depression models based on stress exposure including chronic mild stress (Autry et al., 2011; Pham et al., 2018; Tang et al., 2015), learned helplessness (Koike et al., 2011; Li et al., 2010) and the social defeat stress model (Yang et al., 2016). Conversely, genetic models of depression allow the dissection of specific depressive-like endophenotypes. Specifically, we hypothesized that given ketamine's ability to stimulate glutamate signaling, a genetic model of impaired glutamate function in the PFC can help to identify the molecular mechanisms mediating antidepressant response or resistance to treatment.
Among the three vesicular glutamate transporters (VGLUT1–3), VGLUT1 predominates in the cerebral and cerebellar cortices and hippocampus (Daniels et al., 2004; Wojcik et al., 2004). The latter areas are known to play a key role in integrating affective imprints and cognitive processes. VGLUT1 levels affect glutamate vesicular content and quantal release of glutamate (Daniels et al., 2004; Wojcik et al., 2004). In addition, we have demonstrated depressive-like behavior in a VGLUT1 knock-down model (VGLUT1+/−) (García-García et al., 2009; García-García et al., 2013; Tordera et al., 2007). These mice express half the amount of VGLUT1, and display reduced glutamate release upon stimulation (Wojcik et al., 2004). They also express lower synaptic plasticity amplitudes in long-term potentiation paradigms (Balschun et al., 2010). Also, low VGLUT1 levels may be a biological risk factor for depressive states. Recently, a depression-prone mouse strain created by natural selection of helpless mice showed a downregulation of VGLUT1 in the PFC (Machado et al., 2017). Moreover, different depression models based on chronic stress show VGLUT1 downregulation in cortical areas (Elizalde et al., 2010; Palmfeldt et al., 2016). In line with these studies, clinical data show decreased VGLUT1 levels in the PFC of depressed subjects (Gilabert-Juan et al., 2012; Uezato et al., 2009). Nevertheless, VGLUT1 upregulation could lead to a rise in glutamate function, thus initiating a coordinated cascade that mediates a change in neural plasticity relevant to antidepressant activity (Rapp et al., 2004).
Here, we studied the antidepressant-like effect of ketamine on VGLUT1 knock-out heterozygote mice. The monoamine antidepressant reboxetine was used as a reference drug. Interestingly, we first found that VGLUT1+/− mice were resistant to ketamine. Subsequently, we investigated two pharmacological rescue strategies for the effect of ketamine in these mice. The first strategy involved pretreating the mice with reboxetine to induce a priming effect before ketamine injection. The second strategy consisted of the local expression of exogenous VGLUT1 using recombinant adeno-associated virus (AAV) technology.
Section snippets
Animals
Heterozygote VGLUT1 knock-out (VGLUT1+/−) and wild-type littermate (WT) males C57BL/6 mice (8–12 weeks old) bred in the animal house of the University of Navarra from heterozygous fathers (kindly donated by Dr. S. Wojcik, Gottingen, Germany) and WT mothers (Harlan, France) were used as indicated in supplementary material section. Mice were weaned and genotyped at the age of 3 weeks. Given that mice bred in our animal facility had >30 generations, a genetic background around 99% of C57BL/6N was
Depressive-like phenotype of VGLUT1+/− mice is rescued by chronic reboxetine
Mice body weight was not affected neither by genotype nor reboxetine treatment (data not shown). Repeated measures Two-Way ANOVA with genotype and treatment as between-subject and time (weeks) as within subject factors revealed a significant interaction among treatment and time on sucrose intake (F4,380 = 3.74; p = 0.005). Two-Way ANOVA for each week showed a significant decrease of sucrose intake in VGLUT1+/− mice compared to WT along the 5 weeks measured (main effect of genotype, F1,92
Discussion
This study suggests a key role for PFC VGLUT1 in the modulation of depressive-like behaviors and antidepressant response. Our results show that ketamine fails to trigger rapid antidepressant-like effects and pro-BDNF synthesis in the PFC of the VGLUT1+/− model. Nevertheless, two different strategies aimed at upregulating PFC VGLUT1 in this model rescue the antidepressant activity of ketamine. Further, as has been observed in previous studies in the hippocampus, a specific role for the eEF2
Conclussion
Altogether, through vesicular glutamate transporter type 1 function alterations, we provide evidence that the level of vesicular glutamate stores directly available for release in the PFC is a key determinant of depressive-like behavior and antidepressant action. This finding is relevant for preclinical and clinical evidence that increased glutamate levels underpin the neuroadaptive responses induced by both classic and rapid acting antidepressants.
The following are the supplementary data
Role of funding source
We are very grateful to PIUNA (Universidad de Navarra) and to the Ministerio de Ciencia e innovación (SAF2011-27910) for the financial support of this study. We are also grateful to Asociación de Amigos for the scholarship to Ms. Borja Belloch. Thanks to Ministerio de Ciencia, Innovación y Universidades (Gobierno de España) for supporting to M.C.E (FPU17/05039) with a fellowship. Xao Min Zhang was supported by the Erasmus Mundus ENC program and the labex BRAIN extension grant. Funding from
Author contributions
Francisco de Borja Belloch PhD, performed all the biochemical and behavioral studies directed to characterize the phenotype of the VGLUT1+/− mice under the different pharmacological strategies. He has also carried out the statistical analysis of these studies together with the interpretation. He has been involved in the writing of all the sections of the manuscript.
María Cortés MSc, performed western studies directed to identify the exogenous VGLUT1mCherryminisog expression in the PFC. She has
Ethical statement
This study has been carried out in accordance to the ARRIVE guidelines and have been carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, and the National Research Council's Guide for the Care and Use of Laboratory Animals. The sex of animals has been indicated.
The work is an original study and has not been published previously (except in the form of an abstract or in an academic thesis) and
Declaration of Competing Interest
All the authors declare no biomedical financial interests or potential conflicts of interest.
Acknowledgments
We are very grateful to Sandra Lizaso and Mikel Aleixo and for their excellent technical collaboration in this this study.
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