**4. Discussion**

We show here, for the first time, that following single or repeated exposure, CBD can be found in the rat brain and, specifically, in the medial prefrontal cortex (mPFC). In fact, we found that CBD is present in the mPFC of rats treated either acutely or repeatedly with the phytocannabinoid, with a clear dose −response profile. In fact, whereas CBD cannot be detected at the lowest dose of CBD, i.e., 5 mg/kg, it is measurable at the dose of 15 mg/kg, reaching its peak at the maximal dose tested (30 mg/kg). Similarly, with the brain, a clear dose −response effect of CBD exposure was observed when measuring its levels in the rat plasma. In fact, it is barely detectable at 5 mg/kg of CBD, whereas its concentration increases dose-dependently at the other doses employed, reaching concentrations that are indeed much higher when compared with the brain levels. Again, we observed a significant reduction in CBD following repeated exposure when comparing plasma concentrations with those of single exposure. Taken together, these results indicate that CBD is already bioavailable within 1 h following the single injection and it is still detectable 24 h after the repeated administration, albeit at much lower concentration. Other authors investigated the impact of different methods (pulmonary, oral, and subcutaneous) and dosage of acute administration, on brain and serum level of CBD in rats. For this reason, the comparison among previous works and ours with respect to brain uptake is hard to perform. Independently of the route of administration, doses comparable to those used in our study (10 mg/kg) led to relevant brain uptake. Hlozek et al., showed a peak of concentration of 200 ng/g at 2 h after oral administration that was enhanced by the feeding state of the animals [36]. Another study observed a high brain uptake after acute subcutaneous injection of 10 mg/kg of CBD in mice with a peak reached after 1 h [37]. Again, Deiana et al., evaluated the pharmacokinetics of a high acute dose of CBD (120 mg/Kg) in rats (i. p.), thus measuring a Tmax of 60–120 min and relevant amount of CBD still after 24 h at brain level [38]. To the best of our knowledge, no previous studies regarding repeated exposure to CBD were conducted.

In line with the presence of the phytocannabinoid in the mPFC following a single exposure, we found that a single dose (30 mg/kg) of CBD is sufficient to upregulate Bdnf exon IV, the most abundant variant of the BDNF gene, which is paralleled by a similar increase in cortical mBDNF and TrkB. Such up-regulation drives the selective activation of the PI3K pathway (i.e., Akt). These effects might be beneficial for the overall regulation of cell homeostasis, potentially fostering the ability to promote synaptic transmission and plasticity, neuroprotection, and activity-dependent structural remodeling [14].

Conversely, a slight decrease was observed in the levels of mBDNF paralleled by reduction in TrkB and Akt activation following repeated exposure to CBD in the mPFC. Such a variable profile is not surprising when examining the neurotrophin expression [39,40]. To further strengthen this concept, we have also shown that an opposite modulation of BDNF can dissect the antidepressant from the reinforcing properties of ketamine [41]. These lines of evidence sugges<sup>t</sup> that a more prolonged treatment with CBD should be performed to cause BDNF up-regulation.

As already revised by Lucas et al. [42] and Ujvàry et al. [43], CBD is mainly metabolized by iso-enzymes CYP2C19 and CYP3A4 at hepatic level. Several studies involving rodent models showed that CBD is mainly excreted in the intact or glucuronide form, while the major metabolites are hydroxylated derivatives and their glucuronide conjugates. For this reason, hepatic passage could be involved in the reduction in CBD plasmatic concentration during repeated exposure. However, the pharmacology of hydroxylated metabolites is still poorly investigated.

Overall, these data indicate that a single dose of 30 mg/kg is sufficient to trigger the activation of BDNF and its downstream signaling in the mPFC, whereas repeated exposure to the same dosing regimen downregulates the neurotrophin system. These data highlight the tight dependence of BDNF modulation upon CBD levels in the mPFC. In fact, it appears that when CBD is available in the mPFC in appreciable concentrations, the BDNF system is activated, such as after the single treatment. This finding may also sugges<sup>t</sup> that, at least at

the 30 mg/kg dose, which is indeed not the highest used in the literature, CBD may mediate, through BDNF, some short-term benefits for the brain by facilitating synaptic transmission, rather than long-term benefits. One potential outcome of the increase observed following a single exposure to BDNF relies on the possibility that it may contribute toward setting up a proper adaptive response of neural cells in response to environmental challenges, be they positive or adverse. For instance, we have demonstrated that exposure to stress prevents the ability to mount a neuroadaptive response to adverse external stimuli via up-regulation of cortical BDNF expression [44]. However, it is interesting to note that a single stress also favored the performance in a single cognitive test through transient up-regulation of cortical Bdnf mRNA levels [45]. Based on these lines of evidence, there is the possibility that CBD-induced up-regulation of cortical BDNF expression may favor cell coping under similar situations.

Another interesting finding of our experiments derives from the evidence that BDNF expression is elevated in the rat striatum following repeated exposure. It is well established that the striatum lacks Bdnf messenger RNA and that the neurotrophin is supplied to the striatum through anterograde transport from the mPFC [21]. Notably, we found a reduction in mBDNF in the mPFC and a significant increase in the striatum, potentially supporting an increased anterograde trafficking of BDNF, mediated by CBD. Interestingly, it appears that CBD has also activated the downstream BDNF pathway mediated by Akt, thus mediating the intracellular signaling cascade promoted by the neurotrophin. Notably, it has been previously shown that CBD leads to stronger connectivity between prefrontal cortex and striatum in humans, an effect that may perhaps be due to BDNF trafficking [46]. Evidence also exists that BDNF is critical for the survival of striatal neurons in animal models of Huntington's disease [47]; therefore, repeated treatment with CBD, through BDNF up-regulation, may represent a potential strategy to rescue, at least partially, striatal neurons from degeneration. BDNF is also important for the survival of striatal GABA neurons [48]. This is crucial in view of the notion that dysfunction in cortical and subcortical GABAergic pathways characterize, among others, the pathophysiology of schizophrenia [49]. It is in fact established that, in the striatum, GABA is pivotal for the regulation of overactivity of excitatory neurotransmissions as well as memory functions, which are perturbed in schizophrenic patients [50,51]. Taken together, these data sugges<sup>t</sup> that CBD-induced increase in striatal BDNF may be functionally relevant and may represent an added value for CBD as supplement.
