Here we demonstrate that i.p. CBD and MK-801 injections 30 min prior to extinction training had a common effect of increasing subsequent contextual freezing when contextual fear conditioning was relatively weak. In contrast, CBD replicated the effect of DCS to reduce contextual freezing when conditioning was stronger. These effects of CBD were critically dependent upon the extinction training, as CBD alone had no effect upon subsequent contextual freezing. These results suggest that the long-term impact of CBD on extinction depends upon the prior conditioning experience.

Under the strong conditioning procedure, CBD reduced subsequent freezing by a similar extent to DCS (CBD: 29.5 ± 2.5; DCS: 28.7 ± 9.0). In contrast, MK-801 had no observable effect. Given that DCS has previously been shown to potentiate contextual fear memory extinction (Yamada et al., 2011), this suggests that CBD also enhances extinction. In contrast, and somewhat surprisingly, there was no evidence that MK-801 impaired extinction under the same conditions. While there was no non-extinction control in the MK-801/DCS experiment, the no-extinction control in the CBD experiment suggests that the 20-min extinction session did induce a meaningful reduction in contextual freezing. This is consistent with the literature indicating that DCS only potentiates extinction when there is significant reduction in fear induced by the extinction training itself (Bolkan and Lattal, 2014). Therefore, it remains unclear why MK-801 did not impair this extinction-induced reduction in freezing, especially as it did under the weaker conditioning procedure. Indeed, there was no visual indication of an extinction impairment, as might have been expected with a ceiling effect were the freezing reductions in control animals too modest to observe a significant impairment. Nevertheless, the common effect of CBD and DCS supports the interpretation that each treatment reduced freezing through a potentiation of memory extinction.

While the combination of drug treatment and extinction training to reduce freezing is certainly consistent with an enhancement of extinction, studies of memory extinction rarely rule out alternative interpretations based upon long-lasting direct effects of drug treatment on the test itself. For i.p. injections of DCS, its effect on contextual freezing was not shown to be extinction-dependent (Yamada et al., 2011). However, when infused into the hippocampus, DCS did not have long-lasting direct effects on contextual freezing in the absence of extinction training (Bolkan and Lattal, 2014). Moreover, i.p. DCS reduced contextual freezing only when given before a long extinction session, but increased freezing when the extinction session was shorter (Yamada et al., 2009), demonstrating that these bidirectional effects must be attributable to the nature of the extinction session. In contrast, prior demonstrations of CBD reducing contextual freezing have not shown the effects to be dependent upon the extinction training (Bitencourt et al., 2008; Do Monte et al., 2013). CBD has been shown to have long-lasting antidepressant effects, reducing hyperactivity in an olfactory bulbectomy model of depression in mice (Linge et al., 2016). While these effects were observed with a higher single dose of CBD (50 mg/kg c.f. 10 mg/kg used here), a long-lasting modulatory effect on locomotor activity would likely impact upon the conditioned freezing measure of fear used here. However, in the absence of extinction training, CBD had no impact upon contextual freezing 24 h later. Therefore, CBD likely does potentiate contextual fear memory extinction.

The long-lasting reduction in contextual fear was preceded by an acute effect of CBD to reduce the expression of contextual freezing during the extinction session itself. This acute effect further supports the need for the aforementioned no-extinction controls, in order to rule out any direct persistent effects of CBD to reduce freezing. While CBD has been shown to reduce contextual fear memory expression (Resstel et al., 2006; Lemos et al., 2010) but see (Marinho et al., 2015), in the previous studies of contextual fear memory extinction CBD did not have an acute effect when it did potentiate extinction (Bitencourt et al., 2008; Do Monte et al., 2013). These discrepant effects might be related to the route of administration. Prior extinction studies have infused CBD directly into the infralimbic cortex (Do Monte et al., 2013) or intracerebroventricularly (Bitencourt et al., 2008), whereas the acute fear-reducing effect of CBD appears to involve the prelimbic cortex (Lemos et al., 2010), bed nucleus of the stria terminalis (Gomes et al., 2012), and perhaps the amygdala (Fusar-Poli et al., 2009). Therefore, while direct intracranial infusions dissociate the acute and long-lasting impacts of CBD, systemic injections appear able to induce both effects, presumably via actions in those dissociable neural loci. This is of translational relevance as both acute fear reduction and long-lasting enhancements in fear extinction have clinical benefits.

While the effects of CBD in our strong conditioning procedure are consistent with prior literature, the results from our weak conditioning experiments are less so. Under these conditions, the extinction session appeared to reduce contextual freezing to near baseline levels, such that while MK-801 impaired extinction to increase freezing, DCS did not further reduce freezing. Therefore, we might have expected CBD to replicate the effect of DCS (as it did in the stronger conditioning experiments), resulting in no modulation of contextual freezing. Instead, CBD replicated the effect of MK-801 to increase freezing at test. No-extinction controls confirmed that this elevation in freezing at test was not a direct effect of CBD, as might be predicted from a putative CBD-induced long-lasting attenuation of activity (Linge et al., 2016). Therefore, the effect of CBD is consistent with an impairment in memory extinction. The alternative explanation of an enhancement of reconsolidation is not consistent with the effects of MK-801 and DCS, nor is it supported by previous demonstrations that CBD impairs, rather than enhances reconsolidation (Stern et al., 2012). Why CBD should impair extinction when conditioning is weak is unclear. Although bidirectional memory modulatory effects with the same treatment have been observed previously (Suzuki et al., 2004; Lee et al., 2006), including with strength of conditioning being the only variable (Eisenberg et al., 2003), these have been interpreted as qualitatively similar effects on dissociable mnemonic processes (e.g., impairment of extinction to increase memory expression vs. impairment of reconsolidation to reduce memory expression). A bidirectional effect on extinction based upon the strength of the memory, to our knowledge, has not been previously reported.

There are two possible explanations for why the strength of conditioning should reverse the impact of CBD on extinction: Neuroanatomical competition and the differential stress levels evoked during extinction. In a series of studies of the impact of intra-medial prefrontal cortical infusions of CBD, Guimaraes and colleagues have previously demonstrated opposing effects of CBD depending upon the locus of infusion and levels of stress (Fogaça et al., 2014; Marinho et al., 2015). Intra-prelimbic infusions of CBD reduced the expression of contextual fear conditioning, whereas infusions into the infralimbic cortex increased contextual freezing. Given that our systemic injections of CBD here acutely inhibited contextual freezing during the extinction session following strong conditioning, these conditioning parameters (identical to those used in the infusion studies) might have biased the primary central locus of activity of CBD to the prelimbic cortex. However, while there has been no study to date of the impact of intra-prelimbic CBD infusions upon fear memory extinction, the neuroanatomical underpinnings of fear memory extinction implicates the infralimbic cortex, rather than the prelimic cortex (Sotres-Bayon and Quirk, 2010), consistent with the observation that intra-infralimbic CBD enhances contextual fear memory extinction (Do Monte et al., 2013). Therefore, while the neuroanatomical locus of effect (and indeed also the neurochemical and neurophysiological basis) of systemically-injected CBD in the current study remains to be determined, it is perhaps unlikely that our bidirectional results can be attributed to differential neuroanatomical loci of effects.

The same aforementioned studies of medial prefrontal cortical infusions also revealed apparent effects of stress levels upon the impact of CBD (Fogaça et al., 2014; Marinho et al., 2015). While prelimbic infusions of CBD reduced the expression of contextual freezing, they also increased measures of anxiety in the elevated plus maze (Fogaça et al., 2014). This anxiogenic effect is likely due to the relatively low stress levels induced in the elevated plus maze, as compared to contextual fear conditioning, as when testing was preceded by restraint stress to increase the stress levels present in the plus maze, the impact of CBD was reversed to reduce anxiety (Fogaça et al., 2014). For infralimbic infusions, the pattern of results was largely the reverse of those observed with prelimbic infusions, but restraint stress blocked, rather than reversed, the anxiolytic effect of CBD (Marinho et al., 2015). Although these results are related specifically to the acute expression of fear and anxiety, they reveal the possibility that the longer-term impact of CBD in relation to its effect on extinction might similarly be modulated by the stress levels present during the extinction session. One clear prediction would be that restraint stress prior to extinction following weaker conditioning would block the effect of CBD to impair extinction.

In summary, CBD had bidirectional effects on the extinction of contextual fear conditioning, depending on the nature of the initial fear conditioning. Nevertheless, in the more translationally-relevant stronger conditioning setting, CBD both acutely inhibited fear expression and enhanced extinction to produce longer lasting reductions in fear. These observations provide further support for the potential translational use of CBD in conditions such as posttraumatic stress disorder and specific phobias.