These results indicate the presence of lasting detrimental effects of a chronic treatment with pure THC during adolescence on adult behavior of female rats, thus confirming our previous findings [14,16,22]. In particular, adult female rats chronically exposed to high doses of THC during adolescence display depression- and anxiety-like behaviors, including increased immobility in the FST, anhedonia, reduced social behavior, as well as short-term memory deficits in the NOR test. This is also in line with multiple studies from both humans and animals suggesting that exposure to THC during adolescence has the potential to produce lasting alterations in neurobiology and behavior whose magnitude seems to depend on dosing and timing of THC administration [23]. Notably, research evidence suggests a dose–response relationship for the association between adolescent THC exposure and detrimental effects on brain and behavior, as exposure to THC during adolescence at doses suggestive of a low to moderate dose exposure in humans resulted only in mild or even no long-term deficits in affective or cognitive behavior [24,25].

One of the main criticisms of these studies is that they investigated the effects of pure THC without taking into account the compresence of other cannabinoids in the cannabis plant, which might modulate the overall outcome. In particular, it has been suggested that CBD content in the plant could mitigate some of the side effects of THC, at least in adults [10,26].

In this study, we evaluated the long-term consequences of a concomitant administration of THC and CBD at a ratio of 3, which was chosen based on the analysis of recently confiscated illegal cannabis samples in Europe [18]. The results obtained indicate that the long-term detrimental effects of pure THC were partially prevented when a THC-rich/CBD-poor combination was administered to adolescent female rats. In particular, a protective action of CBD was observed on measures of anxiety-like behaviors. However, depressive-like behavior in the FST, anhedonia and short-term memory deficits in adult female rats were also present following administration of this THC-rich/CBD-poor combination during adolescence. These data suggest that CBD could mitigate some of the long-term adverse effects of THC when exposure occurs in female rats during adolescence, and the protective effect seems to be particularly relevant when anxiety behaviors are considered. We cannot exclude that a lower THC/CBD ratio could result in a more marked protection. Accordingly, when a THC/CBD ratio of 1 was used, in addition to anxiety behaviors, THC-induced cognitive deficits were also prevented, at least in adolescent male mice [27].

In several European countries, a new market for products containing mainly CBD, with very low THC levels (called “light cannabis” or “CBD-rich cannabis”), is emerging. CBD products can be legally distributed if the THC concentration remains below a specified level, ranging from 0.2 to 1% across countries [12]. As there are no regulations on CBD concentration, products with remarkably different CBD concentrations are legally available.

An emerging “light cannabis” market needs to be considered with caution from a public health perspective. As a legal alternative to cannabis with higher THC levels, depending on the users’ appreciation of the psychoactive properties of “light cannabis” products that do not cause the usual “high”, it is possible that a number of young cannabis users may shift to these products. In addition, some teens may initiate use of “light cannabis” products for either recreational or self-medication purposes. In this work, we investigated, for the first time, possible long-term consequences of adolescent administration of a CBD-rich/THC-poor combination at a ratio of 0.33 and with a total THC content of 0.3%, resembling the average contents found in “light cannabis” products available in Italy [19]. Chronic treatment with this CBD-rich/THC-poor combination during adolescence in female rats did not induce depressive-like behaviors in the FST, social behavior deficits nor anxiety-like behaviors in the EPM at adulthood. In contrast, we observed the development of short-term memory deficits and anhedonia in the long term. These data provide scientific evidence for possible long-term adverse consequences of chronic exposure to CBD-rich/THC-poor combinations during adolescence. Although not psychotomimetic, CBD is clearly a psychoactive compound and we speculate that some of its actions might also have detrimental effects on adolescent brain maturation, possibly by interfering with normal developmental trajectories. Accordingly, the same behavioral alterations (i.e., short-term memory deficits and anhedonia) were observed at adulthood after chronic CBD treatment in adolescent female rats.

At the end of behavioral tests, we tried to identify possible neurochemical correlates of the long-term effects triggered by CBD and THC combinations in female rats in comparison with pure THC treatment. We started our investigations based on some of the most consistent long-term changes reported after adolescent THC exposure and associated with the development of THC-induced behavioral alterations: the downregulation of CB1 receptors [15,28], the presence of microglia activation [17], and the alteration of GABA synthesis [16,29]. We focused our analysis on the PFC as previous studies identified this brain region as one of most affected by adolescent THC exposure [15,20]. In addition, brain development continues throughout adolescence, especially in cortical regions, and involves dramatic changes in gross morphology with a loss of gray and a rise in white matter [30,31]. The PFC is known to reach full development late in adolescence, and alterations in its maturation have been implicated in several psychiatric disorders, such as schizophrenia and depression [32]. Indeed, endophenotypes for both these disorders were described in adult female rats exposed to THC during adolescence [14].

Adolescent exposure to THC downregulated CB1 receptor levels in the adult PFC, in line with previous findings [15]. The long-term downregulation of CB1 receptors was prevented when a THC-rich/CBD-poor combination was administered, suggesting a protective effect of CBD also at the biochemical level. No long-term effects on CB1 receptor expression were observed after administration of the CBD-rich/THC-pure combinations.

Activation of microglia within the adult PFC has also been reported as a consequence of adolescent THC exposure [17] and was confirmed by the present results, as demonstrated by the increased expression of the activation marker, CD11b, and changes in Iba1-posive cell morphology, including reduced cell area and perimeter as well as increased soma size and roundness. The administration of the THC-rich/CBD-poor combination increased the expression of the activation CD11b, suggesting an activation of microglia cells. However, the investigation of microglia morphology in these animals provided further information on the activation state of these cells, suggesting that the activation of microglia could be slightly reduced with respect to the one triggered by pure THC. In fact, only alterations in the cell area and perimeter of Iba1-positive cells were present without any changes in soma size and roundness, possibly suggesting an intermediate phenotype of these cells, and thus raising the possibility that the low CBD content present in the preparation could have partially prevented microglia activation. Finally, administration of the CBD-rich/THC-poor combination did not affect microglia activation in adulthood, neither in terms of CD11b expression nor microglia morphology.

In the adult PFC, pure THC treatment during adolescence led to a reduction in the main enzyme responsible for GABA synthesis, GAD67, which was in line with previous findings [16,29] and suggestive of a reduced inhibitory tone in the long term. In contrast, administration of both THC/CBD combinations boosted GAD67 levels above control levels in the same brain region, possibly suggesting a potentiation of GABAergic signaling in the adult PFC. The functioning of the PFC relies on a balance between excitatory and inhibitory neurotransmission. This excitatory/inhibitory (E/I) balance arises from a complex interplay of heterogeneous glutamatergic and GABAergic neurons interacting within a local circuit [32]. In particular, GABAergic interneurons that express the calcium-binding protein parvalbumin (PV) are major regulators of E/I balance and play an important role in prefrontal-dependent emotional and cognitive behaviors [33]. Dysregulations in prefrontal E/I balance were frequently implicated in neuropsychiatric conditions such as schizophrenia, as supported by postmortem analyses that revealed abnormalities in the expression of PV and GAD67 [34]. PFC-related dysfunctions in GABAergic signaling represent a central pathological feature of schizophrenia and seem to be crucially involved in the development of neuronal and behavioral disturbances induced by chronic THC exposure during adolescence [35]. Based on this evidence, it is likely that alterations in GABA synthesis triggered by the administration of different CBD/THC combinations in adolescence could lead to E/I imbalances within the PFC, which might contribute to the cognitive abnormalities observed in these animals. However, a functional evaluation of excitatory and inhibitory transmission is clearly needed to address this hypothesis.