This paper reveals the effects of both antidepressants and drugs with antidepressant-like activity (see “Introduction” section) on the levels of eCBs and NAEs in ex vivo tissue. We examined several brain structures that are either implicated in the pathogenesis of depression (i.e., the prefrontal cortex, frontal cortex, and hippocampus) (Holmes 2008) or linked to anhedonia (i.e., the striatal areas) (Robinson et al. 2012) and are sites of biochemical and morphological changes in depressed patients (Holmes 2008). Additionally, the cerebellum has been recently identified as an area that receives negative functional connectivity from the hippocampus in depressed subjects (Cao et al. 2012). Our results suggest that chronic treatment with antidepressants results in higher levels of AEA in the hippocampus and dorsal striatum along with increased levels of 2-AG in the dorsal striatum. These changes were even maintained after a 10-day drug-free period that followed repeated treatment with ESC and TIA. This is the first study to report alterations in the levels of eCBs and NAEs in the brain after the administration of clinically approved antidepressant drugs (IMI, ESC, and TIA) or drugs with antidepressant-like activity (NAC and URB597).

Some changes in eCBs/NAEs levels could even be observed only 24 h after a single dose the tested drugs. For example, a single dose of either IMI or NAC evoked a significant increase in AEA levels in the hippocampus or dorsal striatum, respectively. Additionally, a single dose of IMI or URB597 increased the levels of 2-AG in the frontal cortex and dorsal striatum, respectively. In contrast, a single dose of either IMI or NAC decreased 2-AG levels in the cerebellum, while ESC and NAC have a similar effect on cortical structures. Administering a single dose of TIA or URB597 resulted in a significant decrease in NAE levels in the hippocampus (PEA and PEA/OEA, respectively), while a single dose of IMI had the opposite effect in this region. Additionally, NAC decreased NAE (OEA) levels in the nucleus accumbens, and ESC decreased NAE levels (both PEA/OEA) in both the frontal cortex and the cerebellum. These changes occurred even though the drugs were rapidly eliminated and both eCBs and NAEs were rapidly degraded. These results imply that acute drug administration can provoke rapid adaptive changes that begin only 24 h after a single dose. Interestingly, these changes were all maintained after chronic administration of these drugs over the course of 14 days with the exception of the increase in hippocampal NAE levels that was observed after a single dose of IMI. Finally, the adaptive changes in the frontal cortex and cerebellum that followed ESC treatment were maintained even after a 10-day ESC-free period.

A potent rise in the levels of eCBs, AEA and 2-AG, was observed in the rat dorsal striatum 24 h after the chronic administration of all tested drugs. In the present paper we also report that striatal eCB levels also increase in response to repeated URB597 treatment. Additionally, withdrawal of this drug for 24 h initiates adaptive changes within the eCB system, which may be associated with the antidepressant-like activity of this FAAH inhibitor. Injecting URB597 2 h before decapitation induced a potent increase in the levels of AEA, PEA, and OEA in multiple brain structures, possibly because it acts in time-dependent manner in which an increase of AEA levels lasts between 30 min and 2 h while PEA/OEA levels are maintained up to 6 h (the present paper; Kathuria et al. 2003; Fegley et al. 2005; Piomelli et al. 2006). A previously study by Bortolato et al. (2007) has suggested that treatment for 5 weeks with URB597 also enhances striatal AEA levels but does not affect 2-AG levels in control rats or rats exposed to chronic mild stress (CMS) (Bortolato et al. 2007). Our findings suggest that the antidepressant drugs may exert their therapeutic effects by normalizing eCB levels within the striatum that have been disturbed during depression. In support of this hypothesis, one cortical symptom of depression is anhedonia, which has been linked to the abnormal functioning of CB 1 receptors in the ventral striatum in rats (Hill et al. 2008b). These same alterations have also been observed in anhedonia-related animal models of depression, including chronic unpredictable stress (CUS) and CMS (Hill et al. 2008b; Reich et al. 2013a, b; Segev et al. 2013). Anhedonia is associated with a weakening of the eCB signal in the ventral striatum and with reduced local levels of AEA (Hill et al. 2008b). In this study we detected changes in eCB levels in the dorsal striatum in response to treatment with IMI, ESC, TIA, NAC, or URB597. In contrast, eCB levels only changed in the ventral region (the nucleus accumbens) after chronic administration of NAC. It is still unclear whether changes in eCB levels directly altered the levels of CB receptors or enzymes, although one previous report indicated that an increase in the density of CB 1 receptors was observed in the ventral striatum after reduced levels of AEA (via increased FAAH activity) occurred in alcoholic suicide victims (Vinod et al. 2010). In this paper, we also report that striatal NAE levels increased after chronic treatment with IMI and NAC. One possibility is that increased PEA and OEA levels could strengthen the effect of AEA on CB or vanilloid (TRPV1) receptors (i.e., the “entourage effect”), which could in turn potentiate the effect of eCBs (De Petrocellis et al. 2001; Smart et al. 2002). Another possibility is that NAEs may increase hippocampal ceramide levels, stabilize mitochondrial function and inhibit the degradation of AEA, which could together have a neuroprotective effect (Skaper et al. 1996; Nagayama et al. 1999). Our findings add to the previous scientific literature regarding the effects of antidepressants on the eCB system with one important contradiction. IMI was previously found to lower the expression of CB 1 receptors in the ventral striatum (Hill et al. 2008b) but had no effect on eCB levels in the rat brain (Bortolato et al. 2007; Hill et al. 2008b). The rise in eCB levels that we observed in this study may be result of either differing IMI dosages (Hill et al. used a lower dose), duration of treatment (Bortolato et al. used a 5-week procedure), and/or the sensitivity of the different methods used to measure eCB levels (isotope-dilution liquid chromatography|[minus]|mass spectrometry vs. LC–MS/MS).

As in the striatum, chronic treatment with antidepressant drugs also enhances AEA levels in the hippocampus. Previous studies have demonstrated that eCB signaling decreases in the hippocampus in an animal model of depression (Hill et al. 2005). Additionally, a reduction in hippocampal size has been observed during depression (McLaughlin and Gobbi 2012), and the local administration of a CB1 receptor agonist in the dentate gyrus has elicited an antidepressant-like response (McLaughlin et al. 2007). The eCB system, particularly AEA/CB 1 receptor signaling, is an important mediator of neurogenesis within the hippocampus. The activation of these receptors directs neural precursor cells into a mitogenic state via the activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which promotes cannabinoid-induced proliferation (Galve-Roperh et al. 2002; Ozaita et al. 2007). Increased hippocampal levels of AEA can both reduce excitotoxic damage within the hippocampus and induce protective mechanisms in hippocampal neurons, which may be linked to the influence of the eCB system on the hypothalamic–pituitary–adrenal (HPA) axis (Marsicano et al. 2003). Thus, rats subjected to chronic stress or repeated administration of corticosteroids experience a drop in both the concentration of eCBs and expression of CB 1 receptors in the hippocampus (Hill et al. 2005, 2008a, 2009). Furthermore, activation of the stress axis, which results from a reduction in the inhibitory effect of hippocampal neurons on limbic structures, has been associated with decreased activation of the eCB system in local GABAergic neurons (Hu et al. 2011). At sites of GABAergic inputs in the hippocampus (CA1 region), the activation of CB 1 receptors induces various mechanisms of synaptic plasticity, including depolarization-induced suppression of inhibition (DSI) and long-term depression of inhibitory synapses (I-LTD) (Lovinger 2008). Additionally, previous studies have suggested that hippocampal levels of 2-AG are elevated 24 h or 10 days after chronic administration of ESC. A recent study found that inhibiting monoacylglycerol lipase (MAGL), which is an enzyme involved in 2-AG degradation, produces antidepressant-like effects through the enhancement of eCB signaling through the mammalian target of rapamycin (mTOR) pathway in the hippocampus (Zhong et al. 2014), which suggests a possible involvement of increased 2-AG levels in the antidepressant mechanism of ESC. In addition to eCBs, NAE levels also change in the rat hippocampus. IMI elicits an increase in both PEA and OEA, while ESC increases PEA levels and NAC increases OEA levels. In contrast, TIA decrease PEA levels, and URB597 decreases both PEA and OEA levels. Along with eCBs, these NAEs may also participate in controlling synaptic plasticity via Kv4.3 potassium channels in hippocampal interneurons along with ascending pyramidal and GABAergic cortical neurons (Burkhalter et al. 2006; Bourdeau et al. 2007). As reported previously, chronic treatment with desipramine (a NA and 5-HT reuptake inhibitor) or tranylcypromine (a monoamine oxidase inhibitor) enhances the expression of CB 1 receptors in the hippocampus, although only tranylcypromine decreased AEA levels in the hippocampus (Hill et al. 2006, 2008c). These studies suggest that the regulation of CB 1 receptors in specific brain structures after antidepressant treatment might result from adaptive changes and could vary depending on the levels of both receptors and ligands. In particular, Bortolato et al. suggested that chronic treatment with URB597 did not increase hippocampal AEA levels; in fact, prolonged (5 week) exposure may instead down-regulate AEA in the hippocampus (Bortolato et al. 2007). However, this effect is still poorly understood.

As reported, there were significant alterations in eCB and NAE levels the rat prefrontal cortex, which participates in a variety of functions including learning and memory. For example, increased activation of the eCB system has been observed to strengthen memory (Lafourcade et al. 2007). Reinforcing emotional memories of aversive stimuli can increase levels of eCBs in the prefrontal cortex, which may induce emotional discomfort during depression. In fact, elevated levels of eCBs and CB 1 receptors have been observed in the dorsolateral prefrontal cortex of alcoholic suicide victims (Vinod et al. 2005). Here, we observed a decrease in the concentration of 2-AG after the chronic administration of ESC and NAC, which may be a potential mechanism for the antidepressant-like activity of these drugs in the prefrontal cortex. In contrast, Hill et al. (2008c) indicated that tranylcypromine increases the level of 2-AG and enhances the density of CB 1 receptors in the prefrontal cortex (Hill et al. 2008c). However, other reports have demonstrated that CB 1 receptors in the prefrontal cortex can participate in the antidepressant actions of CB 1 receptor agonists and that increases in local AEA signaling can modulate stress coping behaviors via the activation of serotonergic neurons in the raphe nucleus (Bambico et al. 2007; McLaughlin et al. 2012). Additionally, chronically administering fluoxetine can fully reverse the enhanced CB 1 -receptor signaling seen in bulbectomized rats (Rodriguez-Gaztelumendi et al. 2009) and can also modulate the function (but not the density) of CB 1 receptors in the prefrontal cortex (Mato et al. 2010). In this study, we also noted an increase in PEA levels in the prefrontal cortex after chronic treatment with IMI, NAC and the FAAH inhibitor. Because PEA administration (5–40 mg/kg) by itself can reduce a mouse’s immobility time in the TST and FST, which is a behavioral indication of antidepressant-like activity (Yu et al. 2011), the increased PEA levels might contribute to the antidepressant-like effect these drugs. In contrast, a decrease in NAE levels was observed in the prefrontal cortex after TIA chronic treatment, and reduced PEA levels were maintained after the TIA-free period. This TIA-specific effect might be related to the adaptive changes associated with NAE depletion or to changes in eCB receptors, enzymes or transporters.

Anti-depressant drugs had different effects in other brain regions. In the frontal cortex, chronic administration of NAC increased AEA levels, while 2-AG levels increased after IMI, TIA and NAC treatment but decreased after ESC treatment. Changes in cortical eCB levels has not yet been established in postmortem or ex vivo studies, although it has been observed that CB 1 receptor density decreases in mood disorders (Koethe et al. 2007) and increases in both depressed suicide victims and animal models of depression (Hungund et al. 2004; Choi et al. 2012). Long-term fluoxetine treatment in obese Zucker rats reduces these elevated CB 1 receptor levels in the frontal cortex, which suggests that the eCB system is involved in mediating the effects of fluoxetine via the influence of 5-HT enhancement on CB 1 receptor levels (Zarate et al. 2008). NAE levels in the frontal cortex also fell both 24 h and 10 days after ESC treatment was withdrawn, which many contribute to the antidepressant effect of ESC via the dampening TRPV1-mediated signaling. In support of this hypothesis, previous studies have suggested that the loss of TRPV1 results in antidepressant, anxiolytic, abnormal social and reduced memorial behaviors (You et al. 2012). However, the exact mechanism remains unclear.

In contrast to chronic IMI treatment, cortical NAE levels were reduced after treatment with TIA and ESC, which most likely stems from their differential effects on NA and 5-HT signaling. One possibility is that the increase in NAE levels observed after IMI treatment might reduce NA release and normalize the increased synaptic availability that is induced by IMI treatment; however, future studies are needed to test this hypothesis.

We also examined the effect of chronic antidepressant treatment on the rat cerebellum, which has recently been implicated in the pathogenesis of depression, specifically disturbances in cerebellar–hippocampal projections (Cao et al. 2012). In this study, we report drug-dependent changes in cerebellar levels of both eCBs (AEA increases after the chronic administration of URB597, while 2-AG decreases after the acute or chronic administration of IMI and NAC and the chronic administration of ESC) and NAEs (PEA increases after the chronic administration of URB597 but PEA and OEA decrease after chronic treatment with IMI or ESC). eCBs act as retrograde messengers in the cerebellum, which allows eCB signals to be transmitted through depolarization of Purkinje cells or local interneurons and permits signal transmission over long distances (Kreitzer et al. 2002). Suarez et al. (2008) detected the presence of components of the eCB system in cerebellar tissue, which suggests that eCBs might participate in the development of cerebellar synaptic plasticity [either long term depression (LTD) or long term potentiation (LTP)] (Suarez et al. 2008). Lowered levels of 2-AG after antidepressant treatment (IMI, ESC and NAC) might regulate the plasticity of synapses being made onto Purkinje cells and could play a key role in normalizing LTD in the cerebellar cortex (Safo et al. 2006; Carey et al. 2011; Zhong et al. 2011).

Interestingly, the effects of antidepressants on the eCB system seem to be short-lived. After a 10-day washout period, eCB concentrations returned to control (vehicle) levels except in animals treated with ESC and TIA. The chronic administration of ESC altered eCB levels in multiple brain regions (e.g., frontal cortex, hippocampus, dorsal striatum, and cerebellum), and these effects were maintained even after the drug-free period. It is still unclear whether adaptive changes existed within the eCB system (e.g., changes in enzyme activity, receptor density, eCB transport, etc.) after 14 days of ESC treatment. However, the drug-free period did increase the levels of NAEs in the nucleus accumbens, which was not observed after the acute or chronic administration of TIA. TIA possesses a unique mechanism of antidepressive action and has a specific pharmacokinetic profile. In fact, recent studies have established that unlike other antidepressants, TIA enhances serotonin reuptake and is not primarily metabolized by the hepatic cytochrome P450 system. TIA also stimulates DA release in the nucleus accumbens and acts as a glutamatergic modulator, which influences central neuronal remodeling and restoration of neuronal plasticity (Invernizzi et al. 1992; Vadachkoria et al. 2009). However, understanding the relevance of these multi-targeted interactions will require further study.