The long‐term effects of prenatal exposure to a cannabinoid agonist on neuro‐behavior in the offsprings are well documented. However, little is known about the effects of prenatal cannabinoid exposure on the embryonic cerebral cortex. This study provides new evidence on how prenatal exposure to a relatively low dose of a synthetic cannabinoid agonist can produce significant effects on developmental processes of the neocortex, such as neuronal migration and differentiation.

Previous studies described CB 1 R expression in the developing rodent cerebral cortex in neural precursors, in early stages of neuronal differentiation and, later, in developing axons (Berghuis et al . 2007 ; Vitalis et al . 2008 ). This study indentified CB1R expression in post‐mitotic migrating pyramidal neurons and interneurons in the rat′s developing cerebral cortex During early stage of corticogenesis (E12.5 and E13.5), CB1R was expressed in radially oriented migrating neurons at the level of the VZ/SVZ, representing early‐born glutamatergic neurons in process of radial migration toward the PP. In addition, we identify CB 1 R expression in tangentially migrating interneurons moving into the superficial migratory stream toward their final positions mainly in the hippocampus. In contrast, Berghuis et al . reported CB 1 R expression in migrating GABAergic interneurons, but only during late stages of corticogenesis as they were undergoing intracortical radial migration (Berghuis et al . 2007 ). On the other hand, Morozov et al . identified a subset of CB 1 R/CCK+ and CB 1 R/reelin/calretinin+ hippocampal interneurons originated in the caudal GE and pallial‐subpallial boundary, but they failed to detect co‐localization of CB 1 R+ cells with interneuronal markers while migrating through the neocortex upon arrival to the hippocampus (Morozov et al . 2009 ). The fact that CB 1 R is expressed in migrating post‐mitotic neurons since early stages of corticogenesis suggests that eCB signaling is required for the regulation of neuronal radial and tangential migration.

Effects of prenatal exposure to WIN on neuronal migration and differentiation in the developing cerebral cortex

The germinative zones of the dorsal pallium also represent transit regions for migrating neurons. In these layers, we found an increase in the number of radial and tangential post‐mitotic migrating neurons in WIN‐exposed embryos. Previous studies have shown that acute treatment with HU‐210, a synthetic agonist of CB 1 R, in organotypic cultures promoted radial migration from the VZ/SVZ to the CP (Mulder et al. 2008). Indeed, CB 1 R and fatty acid amine hidrolase‐deficient mice showed an impaired distribution of cortical pyramidal neurons. In agreement with these findings, we showed that prenatal cannabinoid exposure can impair tangential and radial migration of early‐born post‐mitotic neurons in the developing rat cerebral cortex. Prenatal exposure to WIN also induced an increase in the number of GABAergic interneurons in the superficial migratory stream. Although we observed an increase in the number of DCX+ cells in the VZ/SVZ, we did not find significant effects of prenatal WIN exposure on the number of GABA+ cells in the deep migratory stream. This could be because of the fact that immature interneurons only acquire GABA expression in the course of their maturation and probably a proportion of tangentially orientated DCX+ cell in the SVZ/IZ, still do not express GABA. Since GABAergic interneurons invade the hippocampus mainly throughout a superficial migratory stream adjacent to the MZ (Manent et al. 2006), it would be expected to find a defect in the positioning of hippocampal interneurons in post‐natal brain of WIN‐exposed offsprings. This hypothesis is supported by our present finding of the expression of CB 1 R in migrating DCX+ post‐mitotic neurons tangentially orientated in the superficial pathway. Indeed, prenatal Δ9‐tetrahidrocannabinol‐exposure has been demonstrated to induce an increase in CCK‐expressing interneurons in the early post‐natal rat hippocampus (Berghuis et al. 2005). Berghuis et al. also showed that in vitro pharmacological activation of CB 1 R on CCK‐interneurons induced chemotaxis through a mechanism involving transactivation of brain‐derived neurotrophic factor (BDNF) receptor TrkB. Moreover, it was shown that prenatal WIN‐exposure reduced BDNF levels in hippocampus and frontal cortex of the adult offspring (Maj et al. 2007). Therefore, over‐activation of CB 1 R on developing migrating neurons may interfere in the migratory machinery of a subpopulation of interneurons through CB 1 R and affect their post‐natal positioning and prevent the proper patterning of cortical neuronal networks. Previous evidence suggested that prenatal WIN can permanently alter GABA and Glutamate circuits in the prefrontal cortex and hippocampus (Mereu et al. 2003; Antonelli et al. 2004, 2005). Failure in specification or migration of pyramidal neurons and interneurons could lead to an abnormal distribution into the cerebral cortex and could result in a disturbance of neuronal activity that in turn would be followed by a neurochemical unbalance.

In vitro pharmacological activation of CB 1 R enhanced neural progenitor proliferation (Mulder et al. 2008; Trazzi et al. 2010); however, we did not find significant effects of prenatal cannabinoid exposure on cell proliferation in the embryonic dorsal pallium. This discrepancy could be because of differences in the experimental model, drug administration and/or the duration of the treatment. Nonetheless, the effect of cannabinoids on neural proliferation has not been unambiguously demonstrated in vivo. Therefore, the increase in the number of DCX+ cells in the VZ/SVZ is not because of an increase in cellular proliferation, which in turn reinforces the hypothesis that prenatal WIN exposure interferes with normal neuronal migration.

Although it has been described that chronic exposure to WIN down‐regulates pro‐apoptotic molecules and up‐regulates non‐apoptotic ones in the cerebral cortex of adult rodents (Alvaro‐Bartolome et al. 2010), we did not observe any statistical difference in the number of apoptotic cells in the developing cerebral cortex of WIN‐exposed embryos. Since cell death by apoptosis mainly occurs at early post‐natal ages, during synaptogenis of post‐mitotic neurons, to eliminate neurons which failed to position correctly, it would be expected that any aberrant modulation of apoptotic pathways would be evident at this early ages.

Since Tbr1 is a transcription factor that promotes corticothalamic projection neuron specification (Hevner et al. 2001) and previous studies have shown that CB 1 R inactivation impaired corticothalamic connectivity (Mulder et al. 2008; Wu et al. 2010), we assessed if prenatal WIN exposure had any effect on this neuronal subpopulation. Here, we showed that WIN treatment delayed the differentiation of intermediate progenitor cell (Tbr2+) to post‐mitotic glutamatergic neurons of the deeper layer (Tbr1+), as shown by de increase in the number of Tbr2+ cells and the transient decreased of post‐mitotic Tbr1+ neurons. In line with these results, a recent study revealed that CB 1 R is able to modulate transcription factors that control pyramidal neurogenesis and upper and lower cortical neuron differentiation. CB 1 R‐deficient mice showed a delayed distribution of post‐mitotic Tbr1+ neurons and fate decision changes through the Ctip2 (COUP‐TF interacting protein 2)/Stab 2 (special AT‐rich binding protein 2) transcriptional regulation code (Diaz‐Alonso et al. 2012). With regards to the increase in DCX+ radial post‐mitotic neurons in the SVZ, we could speculate that a delay in glutamatergic neuron differentiation could induce a delay in the onset of migration to the CP. Moreover, prenatal WIN‐exposure induced a transient increased in Cajal–Retzius cells population at early stage of corticogenesis. Therefore, an impaired in Reelin expression could be a factor of deficit in radial migration. On the other hand, another recent work demonstrated that Tbr2+ cortical intermediate progenitors dictate the migratory route and control the amount of GABAergic interneurons in the SVZ (Sessa et al. 2010). Therefore, we could hypothesize that the increase in Tbr2+ cells in WIN‐treated embryos could account for the increase in DCX+ tangential neurons in the SVZ.

In conclusion, this study provides a detailed analysis of the consequences of prenatal cannabinoid exposure in the embryonic cerebral cortex. Alteration in the proper regulation of neuronal specification, differentiation and migration of glutamatergic and GABAergic cortical neurons during cortical development might result in an inappropriate assembly of neuronal networks that could in turn lead to a cognitive and neurobehavioral dysfunction later in adult life. In this context, it is important to note that prenatal WIN‐induced alteration in cortical glutamatergic and GABAergic neurons are associated with a cognitive and neurobehavioral deficit in post‐natal rats (Mereu et al. 2003). The present results contribute to the knowledge on neurobiological substrates that determine neurobehavioral changes that will persist through post‐natal life.