In humans, poor prenatal and early postnatal care is a major developmental stressor commonly found in low socioeconomic groups^1,2. Such stress can affect neurodevelopment during critical periods of early life, greatly impacting both cognitive and behavioral outcomes in adulthood^3–8. Many studies have examined the effects of stress on the cerebrum, such as the hippocampus, and found both neural and behavioral aberrations⁹. However, compelling evidence shows that the cerebellum, which is bidirectionally interconnected with vast regions of the forebrain^10–14, is also profoundly affected by stress^15,16.

Evidence suggests that developmental impairments in humans and rats are mediated by poor regulation of homeostatic systems as a function of early-life stress^17,18. One such homeostatic mechanism is the stress response. Stress typically results in hypothalamic release of corticotropin releasing hormone (CRH), which stimulates the pituitary gland to secrete adrenocorticotropic hormone (ACTH) and, in turn, prompts the adrenal cortex to release cortisol (or the rat homologue corticosterone; CORT). CORT enters the bloodstream and acts on various nervous system targets, which provides negative feedback on the hypothalamic-pituitary-adrenal (HPA) axis, including the hypothalamus. CORT binds to glucocorticoid receptors in hypothalamic paraventricular nucleus (PVN) neurons, a process that ultimately suppresses the HPA axis by inhibiting the continued release of glutamate. The endocannabinoid system plays an important role in this regulation^19,20.

Recent studies indicate that stress may increase risk for the varied detrimental effects of exogenous cannabinoids like Δ9-tetrahydrocannabinol (THC)²¹. Accordingly, it seems imperative to understand the vulnerability that stress imparts on neural systems. Heavily integrated into the stress response system is the endogenous cannabinoid (endocannabinoid) system. Once CORT has activated post-synaptic glucocorticoid receptors, those receptors signal the production of endocannabinoids, such as 2-arachidonoyl glycerol (2-AG) and N-arachidonoyl ethanolamine (anandamide; AEA). Endocannabinoids primarily act in a retrograde manner, signaling at presynaptic receptors to modulate synaptic transmission and the stress response²⁰. CB₁ cannabinoid receptors (CB1Rs) are present at high levels in the prefrontal cortex, hippocampus, and cerebellum, modulating transient and long-lasting forms of synaptic plasticity^22,23 and the stress response²⁴. CB1Rs are more abundant in early life as the endocannabinoid system undergoes significant remodeling and refinement, suggesting that this system is particularly vulnerable during this time of plasticity²⁵.

Just as there are widely known sex differences in the stress response system along the HPA axis, including basal CORT levels, stress responsivity, and glucocorticoid receptors²⁶, there is emerging evidence of sex differences in the endocannabinoid system. For example, in the hippocampus, there appears to be more CB1R protein in males than females, though females tend to show higher CB1R G-protein activation from endocannabinoids, suggesting more efficient coupling of CB1Rs in females²⁵. These sex differences in the cannabinoid system may be important for key sex differences observed in behavior following stressors. Early-life stress affects the endocannabinoid system by (a) decreasing CB1Rs in adulthood in males²⁷ across cerebral regions such as striatum, prefrontal cortex, and amygdala as concluded through [³H]CP55,940 binding and mRNA expression, though findings in females have been mixed²⁸, and (b) increasing gene expression for endocannabinoid degradation enzymes in frontal cortex, striatum, hippocampus, and amygdala²⁹, though surprisingly these effects have not been studied in the cerebellum. Although endocannabinoids have not been thoroughly investigated in the cerebellum, males have been shown to exhibit increased cerebellar 2-AG compared to females³⁰.

Perhaps unsurprisingly, neural regions highly sensitive to stress are also dense with endocannabinoid receptors. The hippocampus, which has received substantial attention in this field, is well known for its association with the formation of declarative and spatial memories. Hippocampal memory formation appears to be highly susceptible to stress, likely due to its numerous inhibitory connections with the HPA axis^31,32. Moreover, the hippocampus manifests one of the highest affinities for cortisol binding^33,34. Human studies indicate that early-life stress is associated with decreased hippocampal volume and activation^5,35,36. Similarly, animal models of stress exhibit dendritic atrophy and impaired stress-hormone receptor binding and expression³⁷ as well as delayed milestones for dentate gyrus development³⁸ in the hippocampal formation following limited bedding. The hippocampus’s role in memory formation and linkage with the HPA axis may explain the array of impairments common to early-life stress and the development of psychopathology³⁹.

Another region dense in CB1Rs is the cerebellum, which has traditionally been understudied in the stress literature. What little evidence exists regarding the sensitivity of this region to stress is compelling. The cerebellum undergoes cellular differentiation even into 2 years of age in humans or 3 weeks in rats, with continued proliferation, development, and reorganization occurring well into adolescence and young adulthood, and is likely vulnerable to early-life experiences during this time⁴⁰.

Research in human populations has indicated that developmental insults, including preterm birth^41,42 and early-life maltreatment^43–45, are related to impairments in cerebellar development, findings which have been corroborated in animal models¹⁵. For example, Llorente and colleagues⁴⁶ found that early-life stress induced cerebellar neuronal degeneration in adult rats, most prominently in males, that experienced this stressor as pups⁴⁶. Wilber and colleagues⁴⁷ revealed an increased number of glucocorticoid receptors within the cerebellar interpositus nucleus after a maternal deprivation early-life stress paradigm that was correlated with a marked behavioral deficit in delay eyeblink conditioning (EBC), a cerebellar-dependent task^47–49. Thus, early-life stress appears to meaningfully impact the cerebellum, its development, and its associated learning processes.

In addition to the interpositus nucleus, EBC requires cerebellar cortex near the base of the primary fissure. Cerebellar cortex is critical for other cognitive functions, creating closed-loop circuits with prefrontal cortex and other cerebral areas that may allow for cerebellum to modulate higher-order functions^50,51. For example, Crus I has been linked to cognitive flexibility⁵², perceptual decision-making⁵³, and social behaviors⁵⁴.

Chronic stress across the life span remodels the hippocampal endocannabinoid system, by increasing 2-AG, which has been linked to increased CORT²⁰. However, despite the abundance of cannabinoid receptors in the cerebellum and clear evidence of its susceptibility to stress, it remains unknown how early-life stress impacts cerebellar endocannabinoids. Further, it has been suggested that neurodevelopmental changes following early-life stress may not be detectable until adulthood^55,56, which brings into focus the importance of studying long-term effects of early-life stress. The current study, for the first time, concurrently examined the effects of early-life stress on endocannabinoids in the hippocampus and cerebellum, both of which are nodes of endocannabinoid signaling and vulnerable to early-life stress.

Here, a naturalistic rat model of maternal stress, limited bedding, was used to test the hypothesis that early-life stress results in long-term changes in the cerebellar endocannabinoid system. Given the amplification of the stress response during this critical early-life period of cerebellar and endocannabinoid system development, as measured by CORT, it was predicted that early-life stress would down-regulate endocannabinoids in adulthood as measured by lipid analysis. Similarly, it was expected that cerebellar cortex and dorsal hippocampus would both show changes in key endocannabinoids, including increased 2-AG and decreased AEA, as has been shown in hippocampal studies of early-life stress, with accompanying changes to cannabinoid-related mRNA. At baseline, females were expected to have lower endocannabinoid and higher CORT levels compared to normally reared males, following previously reported sex differences. Stressed animals were also expected to exhibit impairments in key behavioral processes including recognition memory and social preference, based on previous studies⁵⁷. Moreover, these behavioral differences were not expected to be due to an anxiety phenotype (assessed via elevated plus maze) or gross cerebellar dysfunction (assessed via rotarod). Overall, these predictions form a direct test of the model that early-life stress induces vulnerability of a portion of the cerebellar-endocannabinoid network, with implications for long-term, aberrant behavioral outcomes.

All experimental protocols involving rats were approved by the Indiana University Bloomington IACUC. All methods were carried out in accordance with relevant guidelines and regulations for care and use of laboratory animals.

The current study is the first to our knowledge to investigate the impact of early-life stress on long-term cerebellar endocannabinoid tone in male and female rats. Notable are three major findings: (1) clear baseline sex-specific differences were observed in endocannabinoids and related lipids in a region-specific manner as well as in mRNA expression; (2) developmental stress caused sustained sex-specific changes to CNS endocannabinoid lipid tone with minimal long-term effects on mRNA; and (3) such stress impaired performance on novel object recognition and social preference.

First, baseline sex and region differences in endocannabinoid tone and mRNA expression were found, consistent with previous studies^30,62. Though some studies have found sex differences in cortical mRNA expression²⁹ or cerebellar regional protein differences in males only²², this study, for the first time to our knowledge, identified extensive baseline sex differences in cerebellum. 2-AG was decreased in males compared to females in cerebellar IP (Fig. 4) but increased in cerebellar cortex (Crus I; Fig. 3), whereas LEA was decreased in both regions in males. Within the IP specifically, mRNA expression was significantly different between males and females at baseline (no treatment groups) across a variety of gene systems, emphasizing the robust and broad effects of sex on neural systems.

Such baseline sex differences may cause exogenous cannabinoids to differentially affect the brain in males and females. This serves as a potential explanation for the key differences in outcomes of exogenous cannabinoid exposure in animal^63,64 and human^65–67 models and perhaps also reflects sex differences in use rates^25,66 and adverse side effects^68,69 in humans. Similarly, understanding regional differences in cannabinoids may help predict directional impacts on behaviors emerging from region-specific neural circuits. Thus, baseline differences in endocannabinoid tone, as well as previous work on receptor differences²⁵, may together help make sense of differences in function, at the circuit and behavioral level.

Second, 2-AG was decreased in the cerebellar IP nucleus, but only in limited bedding males compared to normally reared males. A stress-induced rise in 2-AG during a critical developmental period may explain such a finding, resulting in a long-lasting downregulation of the endocannabinoid system to accommodate this new neuronal environment. In addition, a decrease in AA and a trending decrease in AEA was noted, suggesting that an upstream mechanism may be implicated in these effects. For example, down-regulation could be the function of a substrate imbalance, such as a decrease in phospholipids resulting in decreased overall endocannabinoid production. Although no mRNA changes for these enzymes were found, expressed protein or enzyme activity were not evaluated and, thus, this hypothesis cannot be ruled out.

No endocannabinoid changes were observed in stressed compared to non-stressed females in the cerebellar IP nucleus, though stressed females did exhibit a significant increase in prostaglandin E₂ (PGE₂). PGE₂ has been shown to increase cerebellar Purkinje cell arborization via estradiol production^70–72. Estradiol- and PGE₂-induced cerebellar Purkinje cell arborization may function as a neuroprotective mechanism in females, perhaps further explaining the limited behavioral impairments observed in females during stress paradigms^73–75. Further, the increase in PGE₂ suggests activation of cyclooxygenase (COX) enzymes, which may be related to an increase in pro-inflammatory cytokines, identified as a neural response to stress to activate microglia^72,76. Similarly, the increase in N-acyl glycines in stressed females suggests a pro-inflammatory response, as N-acyl glycines have been implicated in microglial migration^76–78. In fact, microglia have been identified as having a critical role in the sexual differentiation of the brain⁷⁹.

Early-life stress induced the opposite sex-specific profile in the cerebellar cortex (Fig. 3) compared to the cerebellar IP nucleus (Fig. 4): 2-AG was downregulated in stressed females compared to control females in Crus I of cerebellar cortex, whereas in cerebellar IP, 2-AG was downregulated in stressed males compared to control males. Cerebellar behaviors, such as associative learning, require carefully coordinated communication between the cerebellum’s nuclei and cortex. Sex-specific differences in circuit coupling resulting from endocannabinoid regulation of neuronal activation may contribute to hypotheses of baseline and stress-effects on behavioral performance differences in cerebellar tasks, like eyeblink conditioning^47,80,81. Understanding regional specificity within the cerebellum is critical, as extensive work has shown that distinct cerebellar cortical regions exhibit differential functional connectivity with cerebrum in humans and animals^52,82. Though this study did not investigate differences across cortical regions, the question becomes increasingly important as we begin to understand more about how cerebellar signaling projects outwards through the deep nuclei to regulate cerebral signaling (cf.⁵¹) and consider the importance of transmitter systems, such as endocannabinoids, in modulating these processes.

Many studies looking at stress-related endocannabinoid changes have investigated the early effects of stress, finding increased 2-AG and decreased AEA^20,83,84. Thus, the current findings do not preclude the possibility that an acute change in endocannabinoid tone occurred immediately after the stressor; rather, the current study may suggest that any changes taking place close to the occurrence of the stressor were not enduring in these regions. Such changes may not have been maintained due to the early development of the rat hippocampus and lack of continual stressors throughout the study⁸⁵.

Like 2-AG, PGE₂ showed a region-by-sex effect: upregulated in female cerebellar interpositus nucleus following stress but upregulated in stressed males in the hippocampus. As detailed above, such findings may relate to a potential pro-inflammatory response in male hippocampus following stress. Alternatively, these findings reinforce theories of foundational effects of stress on development and sexual differentiation of the brain⁸⁶ particularly for males, further reflected by baseline regional and sex differences in the endocannabinoid system and transcriptomics generally.

By adulthood (P70), male rats having undergone stress exhibited an increase in basal systemic CORT, suggesting the stressor carries long-term effects. These findings are in line with a growing literature that males are more sensitive to stress throughout development due to sex-differences in the uterine environment⁸⁶. In females, increased CORT was not observed, perhaps due to a resilience or compensatory mechanism contributing to female pups overcoming insults to the broader HPA system, but this resilience is lacking in the still-developing endocannabinoid system. In humans, changes in cortisol have been linked to differences in cannabis use⁸⁷; the increased plasticity of the HPA system may explain sex-differences in exogenous cannabinoid use and effects.

Taken together, within a given neural region, stressed males exhibited a similar lipid profile to non-stressed females. Such relationships have been previously shown in the literature and lend support to changes in estrogen receptors, perhaps explaining this feminization of neural circuits susceptible to stress in the male brain⁸⁸.

A third major finding is that early-life stress was associated with functional outcomes, particularly NOR performance, irrespective of sex. As shown in other studies of limited bedding⁹ or maternal separation^89,90 and in the current study, rats that underwent limited bedding stress exhibited recognition memory deficits compared to controls. This was not the case for social preference, which was only impaired in stressed females in the current study. Other studies of early-life stress have shown that males are affected in social behavior later in life^91–95. One reason for this discrepancy may be that Crus I stress-effects on lipids were specific to females. Interestingly, a recent study by Stoodley and colleagues⁵⁴ found that right cerebellar Crus I disruption was related to social impairments in mice. Thus, cerebellar signaling disruption in females may have contributed to this behavioral phenotype.

The current study provides a necessary springboard to understand sex-specific, developmental vulnerability mechanisms conferred by stress, specifically those that provide long-lasting neural changes. With increased interest in the cerebellum’s role as a node of cognitive processing^10,100 as well as its role in psychopathology, such as autism^101,102 and schizophrenia¹⁰³, this line of inquiry is timely and impactful. Understanding these sustained vulnerabilities may help reveal mechanisms of risk, including two-hit models of the effects of early stress and adolescent or adult cannabis use^21,64, and may also contribute to better-informed animal models of exogenous cannabinoid effects.

The current work has been funded by the NIMH (F31 MH119767 to ABM, T32 MH103213 to ABM and WPH, Indiana CTSI Fellowship Award TL1 TR002531 and UL1 TR002529 to ABM, R01 MH074983 and R21 MH118617 to WPH and BFO) and NIDA (K05 DA021696 and R01 DA043982 to KM, R01 DA048012 to WPH and BFO, RO1 DA041208 to HBB, and T32 DA024628 to EL). RNA sequencing studies were carried out in the Center for Medical Genomics at Indiana University School of Medicine, which is partially supported by the Indiana Genomic Initiative at Indiana University (INGEN); INGEN is supported in part by the Lilly Endowment, Inc. We wish to acknowledge Dr. Andrea Hohmann and her lab for equipment use and training, including the rotarod and elevated plus maze. Additionally, we thank Jim Wager-Miller of the Mackie Lab for his training and assistance in study procedures. We also thank the IU Bloomington animal veterinary and care staff. Finally, we thank the engineering staff of the IU Psychological and Brain Sciences department, especially Jesse Goode, Rick Moore, Cameron Walker, and Allen Cody, for assistance building the apparatus for this study as well as helping set up recording equipment for behavioral tasks.

A.B.M., W.P.H., K.M., B.F.O., H.B.B. and J.T.G. contributed to project conceptualization and data interpretation; A.B.M., E.R.L., A.G., E.L. and L.A.B. collected, processed, and analyzed the data; A.B.M., E.R.L. and A.G. substantially drafted the manuscript; and all authors reviewed and provided substantive revisions of the manuscript.

The authors declare no competing interests.

is available for this paper at 10.1038/s41598-020-64075-4.