Adolescent THC exposure altered the developmental trajectory of dendritic arbors

To investigate the morphological consequences of adolescent THC exposure, we reconstructed the apical and basal dendritic trees of layer III PrL pyramidal neurons (Fig. 1d). Arbor development was markedly reorganized by drug exposure. There was no difference in total dendritic length in apical or basal trees at either time point (Supplementary Fig. 1), but Sholl analysis detected interactions in apical (treatment×time×Sholl interval interaction: F 15,420 = 2.1, P = 0.009, Cohen’s d = 0.76–1.19 for significant pairwise comparisons) and basal (treatment×time×Sholl interval interaction: F 7,196 = 2.71, P = 0.01, Cohen’s d = 0.98–1.15 for significant pairwise comparisons) trees indicating that development and THC exposure altered dendritic arbor complexity (Fig. 2a–g, Supplementary Video 1). Across development, VEH-treated subjects exhibited stable apical trees (Fig. 2c) but expanded complexity of basal trees into adulthood (Fig. 2f). This pattern differed significantly for adult rats with a history of adolescent THC exposure insomuch that this treatment increased arborization in distal apical and basal trees 24 h after the last injection (Fig. 2a, d). In early adulthood, however, 2 weeks after drug exposure, THC-treated animals showed significant atrophy in distal apical arbors beginning at 270 μm from the soma and increased proximal apical arborization between 60 μm and 120 μm from the soma (Fig. 2b). Moreover, only THC-treated rats exhibited a reduction in basal tree complexity from adolescence to adulthood (Fig. 2f) emphasizing the marked reorganization of arbor development induced by drug exposure.

Fig. 2 Adolescent THC exposure altered the dendritic arborization, spine density, and developmental trajectory of layer III prelimbic (PrL) pyramidal neurons. a Adolescent THC significantly increased distal apical dendritic arborization in early adolescence (24 h after drug treatment). b In early adulthood (2 weeks after drug treatment), a significant allostatic reversal resulting in atrophy was observed in distal apical trees accompanied by the emergence of significantly increased proximal branching. c Between time points, vehicle-treated animals (VEH) exhibited no difference in apical arbor complexity while THC-treated animals (THC) exhibited a significant decrease in distal apical branching complexity. d Adolescent THC exposure significantly increased basal dendritic arborization 24 h after drug treatment. e Two weeks after drug treatment, a non-significant allostatic reversal was observed in basal trees. f Vehicle-treated animals exhibited significantly increased basal arbor complexity between time points, whereas THC-treated animals exhibited significantly decreased basal apical branching complexity. g Representative dendrograms of traced PrL pyramidal neurons. h No significant differences were observed in total apical dendritic spine density. i Vehicle-treated subjects exhibited significant pruning of stubby apical dendritic spines between time points, whereas THC-treated subjects exhibited premature pruning. j No significant differences in apical mushroom spine density were observed. k No significant differences in apical thin spine density were observed. l Vehicle-treated subjects exhibited significant pruning of total basal dendritic spines between time points, whereas THC-treated subjects exhibited premature pruning. m Vehicle-treated subjects exhibited significant pruning of stubby basal dendritic spines between time points, whereas THC-treated subjects exhibited premature pruning. n No significant differences in basal mushroom spine density were observed. o No significant differences in basal thin spine density were observed. p Representative deconvolved basal dendritic branches from all treatment conditions. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Data shown as mean + SEM a–b, d–e, h–o. Data represent the difference between 2 weeks and 24 h, shown as mean + standard error of the difference between treatments c, f. Scale bar = 2 μm p Full size image

Adolescent THC exposure prematurely prunes dendritic spines

In addition to the complexity of dendritic trees, the spines on arbor branches are a central morphological feature of synaptic plasticity involved in the maturation of the cortex and implicated in the pathophysiology of neuropsychiatric disorders [42, 43]. We investigated the consequences of adolescent THC exposure on dendritic spines by reconstructing branches of dendritic arbors and quantifying the type—mushroom, stubby, or thin—and density of dendritic spines (Fig. 1e). Only stubby spines showed significant developmental fluctuation and sensitivity to THC exposure. VEH-treated animals exhibited reduction in apical stubby spine density (Fig. 2h–k) between adolescence and adulthood (t 15 = 3.90, P = 0.001, Cohen’s d = 1.78) consistent with pruning normally seen during this developmental period. This pattern was absent in THC-treated rats (t 9 = 0.27, P = 0.268, Cohen’s d = 0.72). Instead, reduced stubby spine density was already evident in THC-treated rats compared with VEH-treated rats 24 h after the last injection (t 11 = 2.35, P = 0.039, Cohen’s d = 1.31) and those levels remained low into adulthood. A similar pattern was observed in basal arbors (Fig. 2h–p), which showed a marked reduction in stubby spine density between time points in VEH-treated (t 15 = 4.82, P < 0.001, Cohen’s d = 2.37), but not THC-treated (t 9 = 1.29, P = 0.228, Cohen’s d = 0.78), animals. These findings suggest a morphological correlate of adolescent THC-mediated alterations of PFC structure observed in humans [14,15,16].

Analysis of PrL pyramidal neuron and non-pyramidal cell transcriptome

Based on the morphological impairments induced by THC, we next wanted to expand insights about the molecular make-up of the PrL cells. As such, we developed a strategy to integrate layer-specific LCM and next-generation RNA-seq to measure the transcriptome of morphologically distinct layer III cellular populations (Fig. 1f). We confirmed cell type specificity by capturing and sequencing morphologically identified pyramidal and non-pyramidal populations (Fig. 3a). Sequencing revealed 9740 genes that met criteria for detection. Between these distinctively captured cellular populations, 327 (3.4%) genes were significantly enriched in pyramidal population, whereas only 187 genes (1.9%) were enriched in the non-pyramidal populations, presumably reflecting the latter fraction’s more heterogeneous composition (Fig. 3b, Supplementary Table 1). When holistically comparing the enrichment pattern of the LCM-derived pyramidal population to sorted mouse neocortical neurons (Fig. 3c, Supplementary Table 1), there was a statistically significant linear regression between the fold differences (r2 8236 = 0.126, P < 0.0001) that was especially pronounced when only differentially expressed genes are included in the analysis (r2 461 = 0.442, P < 0.0001). Among these, consistently differentially expressed genes included Neurod6, which was enriched in the pyramidal population, and Lrp4, which was enriched in the non-pyramidal population (Fig. 3d) [44]. In silico cytometry, performed using CIBERSORT, confirmed that the pyramidal population was significantly more neuronal than the non-pyramidal population (t 2 = 6.67, P = 0.022), the latter being an admixture of neuronal and glial components (Fig. 3e). Furthermore, non-linear cluster analysis using t-SNE highlighted the topographical relatedness between the pyramidal population and human excitatory neocortical neurons (Fig. 3f), especially cortical projection neurons from layers II and III (Ex1 subtype) defined by Lake et al. [45]. On the other hand, this analysis highlighted that the non-pyramidal population was more related to inhibitory cortical neurons and non-neuronal cortical cells (Fig. 3f). Overall, these results validated the technical approach by highlighting this strategy’s ability to characterize cell- and layer-specific transcriptional landscapes.

Fig. 3 Laser capture microdissection (LCM) and RNA-sequencing of pyramidal neurons and non-pyramidal cells. a Representative micrographs highlighting Nissl-stained frozen tissue (top) and NeuN immunohistochemistry (bottom). Arrows indicate pyramidal neurons and arrowheads indicate adjacent non-pyramidal, NeuN- cells. b Scatter plot displaying genes expressed and statistically enriched in pyramidal (purple) and non-pyramidal (orange) populations (P < 0.01). c Scatter plot displaying relationship between pyramidal enrichment pattern (x axis, LCM) and neuronal enrichment (y axis, sorted mouse neocortex [44]). Thin line indicates regression with all genes and thick line indicates regression of with only differentially expressed (colored) genes. Counts in each corner indicate number of genes within the respective quadrant that were both statistically enriched (P < 0.01 based on LCM) and showed at least a 22-fold enrichment pattern (based on sorting). d Data from Zhang et al. [44]. highlighting the enrichment pattern of Neurod6 and Lrp4, enriched in neuronal and non-neuronal cells, respectively. e In silico cytometry revealed enrichment of neuronal cells within the pyramidal population and a relative depletion of this cell type in the non-pyramidal population (P < 0.05). f Non-linear cluster analysis highlighted the topological relatedness between the pyramidal population (purple) and excitatory cortical neurons (blue) from the human neocortex, especially the Ex1 subtype (dark blue), which reflects layers II and III cortical projection neurons as defined by Lake et al. [45]. The non-pyramidal cells (orange), on the other hand, were more similar to glia (green) and inhibitory cortical neurons (red) Full size image

Short-term and protracted effects of adolescent THC on the transcriptome of PrL pyramidal neurons

After confirming our capacity to selectively isolate and sequence morphologically defined PrL cellular populations, we interrogated the transcriptomic profile of layer III PrL pyramidal neurons in the rat adolescent THC model. Sequencing revealed 12,568 genes that met criteria for detection and in silico cytometry for each animal revealed that the microdissected material derived predominantly from neurons (93.5 ± 0.6%), suggesting minimal contamination from non-neuronal sources (6.5 ± 0.2%) with no difference between the groups (all P > 0.05; Supplementary Fig. 2).

Evaluation of the adolescent and young adult time points identified genes associated with the early and protracted effects of THC, respectively. As compared with VEH-treated animals, 698 (5.6%) differentially expressed genes were identified in the THC-treated animals 24 h after their last injection (Supplementary Fig. 3a, Supplementary Table 2). Gene enrichment analysis revealed that these genes related to cellular response to organonitrogen compound and Cul3-RING ubiquitin ligase complex (Supplementary Fig. 3a). In early adulthood, 2 weeks after the animals’ last injection, 608 (4.8%) differentially expressed genes were identified in THC-treated animals compared with controls (Supplementary Fig. 3b, Supplementary Table 2). In contrast to the early changes, THC had protracted effects on gene networks associated with microtubule organization and cytochrome complex assembly (Supplementary Fig. 3b).

Adolescent THC alters the normal developmental trajectory of the PrL pyramidal transcriptome

Based on the altered trajectory of morphological complexity seen after adolescent exposure to THC, a key question was whether THC exposure affected the transcriptional ontogeny of PrL neurons. Across development, from adolescence to early adulthood, 797 (6.3%) genes were significantly different in the adult VEH-treated animals when compared with the younger control animals (Fig. 4a, Supplementary Table 2). Instead, 975 (7.8%) genes were significantly different in the adult THC-treated animals (Fig. 4b, Supplementary Table 2) when compared with the younger THC-treated animals. Relative to the biological processes engaged by normal development, genes dysregulated by developmental THC uniquely disrupted epigenetic mechanisms (Fig. 4c). Specifically, VEH-treated animals exhibited changes in genes associated with signal transduction, cytoskeletal protein actin projection protrusion (lamellipodium), and cell morphogenesis, all as expected during normal development (Supplementary Fig. 4), whereas THC-treated animals exhibited changes in genes associated with, not only actin cytoskeleton and dendritic regulation, but also chromatin modification and histone methylation (Fig. 4c). Surprisingly, there was little overlap in these developmentally dynamic genes, with only 83 (4.9%) shared between the VEH- and THC-treated developmental contrasts (Fig. 4c). Within the pathways dysregulated by developmental THC, top differentially expressed genes, replicated using NanoString (Supplementary Fig. 5, Supplementary Table 3), included Dstn, Pacsin1—which was highly enriched in the pyramidal fraction (log 2 FC = 6.17, P = 0.006, Supplementary Table 1)—and Bap1 (Fig. 4d). These data demonstrate that cannabinoids profoundly disrupt the developmental trajectory of layer III PrL pyramidal neuron’s transcriptome.

Fig. 4 Effect of normal development and THC exposure on transcriptional landscape. a–b Development in vehicle-treated animals a resulted in 797 differentially expressed genes, whereas development in THC-treated animals b resulted in 975. Genes differentially expressed in both contrasts are colored blue (similar direction) or red (opposite direction), whereas other colors correspond to the biological categories shown in c. c Genes differentially expressed by developmental THC, which minimally overlapped with those differentially expressed in VEH-treated animals c’, engaged biological processes related to development, chromatin organization, and metabolism, as well as pathways related to cytosolic and nucleolar components. d Representative genes from these pathways included Dstn (top, actin cytoskeleton), Bap1 (middle, chromatin organization), and Pacsin1 (bottom, developmental processes), whose differential expression were replicated using NanoString (indicated by vertical bars with RNA-seq data shown as vertical lines for comparison). e Epigenetic- and dendritic-related genes within the WGCNA green-yellow module were significantly co-expressed in the THC-treated animals (THC) insomuch that a subset of genes were positively correlated, whereas a majority were anticorrelated. This pattern of coordinated expression was, however, not observed in the vehicle-treated animals (VEH). e Differential expression of the epigenetic-related genes from the WGCNA green-yellow module. *P ≤ 0.05, relative THC at 24 h. Data shown as mean ± SEM Full size image

Marked disturbances associated with histone modification and chromatin remodeling were observed across development in THC-treated animals (Supplementary Table 4). Enrichment analyses of the differentially expressed genes indicated the strongest functional association with Kmt2a (also known as Mll1) and histone H3 lysine 4 trimethylation (H3K4me3). Interestingly, Kmt2a—a chromatin methyltransferase—mediates its activity specifically at H3K4 and is highly implicated in cellular processes linked to neurodevelopment and psychiatric disorders [46].

To assess the developmental effects of THC on coordinated transcriptional expression, WGCNA on the RNA-seq data was performed. Developmental THC was significantly associated with one module (dark-grey, P = 0.012), whereas four other modules were developmentally regulated (green-yellow, P = 8.5 × 10−4; tan, P = 7.79 × 10−3; green, P = 0.0082; skyblue, P = 0.027). Pathway analysis (MetaCore; Thomson Reuters) of the most significant developmentally regulated module—green-yellow—revealed significant enrichment of genes related to organelle organization (103 genes, P = 1.18 × 10−11), cellular component organization (146 genes, P = 2.38 × 10−11), chromatin modification (33 genes, P = 9.84 × 10−11) and histone modification (24 genes, P = 2.63 × 10−9; Supplementary Table 5). Interestingly, several genes within this module were significantly affected by THC across development including the histone acetyltransferase Jade2 (log 2 FC = 5.65, P = 2.2 × 10−5), histone arginine methyltransferase Prmt5 (log 2 FC = 1.66, P = 0.013)—which was highly enriched in the pyramidal fraction (log 2 FC = 6.82, P = 0.001, Supplementary Table 1)—and transcription factor Cebpg (log 2 FC = −1.43, P = 0.002).

We further investigated the co-expression between epigenetic modifiers and dendritic regulators within the green-yellow module (23 and 17 genes, respectively) to identify THC-dependent interactions between these two relevant categories. Using non-parametric Spearman’s rank correlation, 30.02% of all possible gene-gene pairs showed significant correlations (at P < 0.05) in the THC-treated animals compared with only 5.95% in the controls (Fig. 4e), suggesting a strong functional relationship between epigenetic mechanisms and dendritic morphology specific to developmental THC exposure. Indeed, substantially more chromatin modifiers from this module were developmentally regulated in THC-treated animals (7/23 genes) than in controls (1/23 genes), again indicating a strong THC-related impairment of epigenetic regulation during development (Fig. 4f).

Developmentally regulated THC gene networks overlap those in schizophrenia subjects

Given the relationship between cannabis use, PFC development, and schizophrenia, we evaluated the relationship between genes dysregulated by developmental THC and human schizophrenia. We interrogated the CMC sample data set consisting of 537 subjects (258 subjects with schizophrenia and 279 controls) with dorsolateral PFC RNA-seq data [41]. Comparing developmentally dysregulated genes (based on differential gene expression analysis) to genes coregulated in schizophrenia (termed schizophrenia CMC modules), genes developmentally dysregulated by THC overlapped significantly with the red (OR = 1.92, P = 9.50 × 10−4), turquoise (OR = 1.42, P = 3.05 × 10−3), cyan (OR = 1.82, P = 0.012), blue (OR = 1.47, P = 0.012), yellow (OR = 1.43, P = 0.028), and tan (OR = 1.57, P = 0.048) schizophrenia CMC modules (Fig. 5a, upper heatmap, Supplementary Table 6). The top three schizophrenia CMC modules—red, turquoise, and cyan—were especially noteworthy as they each significantly overlapped with multiple developmental THC co-expression modules (identified in WGCNA), further suggesting that developmental THC dysregulates PFC expression of schizophrenia-associated genes (Fig. 5a, center heatmap, Supplementary Table 6). In support of this overlap analysis, these three schizophrenia modules were significantly enriched for genes involved in retrograde eCB signaling, glutamatergic signaling, as well as axonal guidance, synaptic transmission, and neuroplasticity (Supplementary Fig. 6). Interestingly, the red, turquoise, cyan, and blue schizophrenia CMC modules were the most dysregulated networks among cases with schizophrenia (Fig. 5a, lower heatmap) [41]. These data highlight similarities in network perturbations owing to early THC exposure and schizophrenia.

Fig. 5 Genes developmentally dysregulated by adolescent exposure to THC are also dysregulated in the DLPFC of humans with schizophrenia. a Enrichment analysis shows significant overlap between genes differentially expressed by developmental THC and co-expression modules identified in human schizophrenia. In addition, these schizophrenia CommonMind Consortium (CMC) modules overlapped with developmental THC co-expression modules further suggesting a common epigenetic landscape. b Pathway analysis was performed on an overlapping set of 181 genes to further explore the biological relevance of these shared genes. c Ingenuity pathway analysis revealed that these genes are predicted to enhance cytoskeletal function (organization and formation) and suppress neurite branching in the THC-treated animals, but not in the vehicle-treated animals (see Supplementary Fig. 7), based on differential expression Full size image

To identify shared sets of co-expressed genes in an agnostic fashion, our subsequent pathway analysis focused on the genes common to the overlapping schizophrenia and developmental THC modules (at P < 0.01). From the 181 genes that met criteria (Fig. 5b), there was a significant enrichment for cytoskeletal and neurite development, which were predicted to be activated and deactivated, respectively (Fig. 5c). The predicted deactivation in neurite development is particularly noteworthy given that animals exposed to developmental THC exhibit decreased dendritic branching. In control animals, on the other hand, neurite branching was activated (Fig. 5c inset, Supplementary Fig. 7) consistent with the increased dendritic branching observed in normal animals during the developmental window studied.