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Cannabidiol has a unique effect on global brain activity: a pharmacological, functional MRI study in awake mice

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Associated Data

All data can be accessed through a link to Mendeley. DOI to follow.

Abstract

Background

The phytocannabinoid cannabidiol (CBD) exhibits anxiolytic activity and has been promoted as a potential treatment for post-traumatic stress disorders. How does CBD interact with the brain to alter behavior? We hypothesized that CBD would produce a dose-dependent reduction in brain activity and functional coupling in neural circuitry associated with fear and defense.

Methods

During the scanning session awake mice were given vehicle or CBD (3, 10, or 30 mg/kg I.P.) and imaged for 10 min post treatment. Mice were also treated with the 10 mg/kg dose of CBD and imaged 1 h later for resting state BOLD functional connectivity (rsFC). Imaging data were registered to a 3D MRI mouse atlas providing site-specific information on 138 different brain areas. Blood samples were collected for CBD measurements.

Results

CBD produced a dose-dependent polarization of activation along the rostral-caudal axis of the brain. The olfactory bulb and prefrontal cortex showed an increase in positive BOLD whereas the brainstem and cerebellum showed a decrease in BOLD signal. This negative BOLD affected many areas connected to the ascending reticular activating system (ARAS). The ARAS was decoupled to much of the brain but was hyperconnected to the olfactory system and prefrontal cortex.

Conclusion

The CBD-induced decrease in ARAS activity is consistent with an emerging literature suggesting that CBD reduces autonomic arousal under conditions of emotional and physical stress.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-021-02891-6.

Keywords: Tonic immobility, Behavioral arrest, Reticular activating system, Olfaction, N-acyl-phosphatidylethanolamines-specific phospholipase D, PTSD, Negative BOLD

Introduction

CBD has anxiolytic properties, reducing the autonomic and emotional responses to stress and interfering with the consolidation and extinction of fearful memories [1], which has been associated with anxiety disorders [2], autism spectrum disorder [3], psychosis [4] and post-traumatic stress disorder [5]. It’s potential as a therapeutic compound is emphasized by the fact that CBD is the primary active compound in the anti-epileptic drug, Epidiolex [6]. CBD has a complex pharmacology within the brain impacting multiple receptors by altering the lipidome, increasing and/or decreasing lipid mediators in specific brain areas [7], associated with dose, neurological condition and the environment. The primary targets for CBD given systemically are unknown. Non-invasive magnetic resonance imaging (MRI) using changes in BOLD (blood oxygen level dependent) signal has been used to detect the immediate increases and decreases in site-specific brain activity in response to various drugs [8–11]. The changes in BOLD signal are basically a proxy for increases and decreases in cerebral blood flow to areas of increased and decreased metabolic activity, respectively. Several studies in humans have used functional BOLD imaging to look at the neuroanatomy affected by treatment with CBD [12–19]. These studies looking at the effects of CBD have all evaluated a single oral dose given prior to scanning. While this approach establishes a baseline of resting state blood flow that changes with different task-related paradigms or differs from placebo or healthy controls in response to a preexisting condition, they do not address the effects of repeated exposure or the potential for dose-dependent changes in activity, consistent with drug target specificity.

Pharmacological MRI (phMRI) is a non-invasive method to evaluate neural circuitry involved in the behavioral effects of drugs independent of their specific biochemical mechanism [20]. To our knowledge, no published reports, in either animals or humans, have used phMRI to assess the immediate dose-dependent effects of CBD on global brain activity. Therefore, the goal of the present study was to characterize the dose-dependent changes in brain activity induced by CBD. Given that CBD may have a narrow dose range, impact multiple targets, and show context-dependent efficacy, phMRI is an ideal method to globally assess the integrated effects of CBD across multiple neural circuits to understand how CDB may impact anxiety and fear. We predict that at a certain dose there would be a decrease in relative activity, assessed using BOLD, in neural circuitry controlling stress-related behaviors. To test our prediction, we imaged awake mice using three different doses of CBD.

Methods

Animal usage

Male C57BL/J6 mice (n = 60), ages 100–120 days, weighing between 28–30 g, were obtained from Charles River Laboratories (Wilmington, Massachusetts, USA). While a majority of phMRI studies have been conducted in rats [21], we chose to study mice based on previous work from our group on CBD induced changes in N-acyl-phosphatidylethanolamines-specific phospholipase D (NAPE-PLD) activity [22]. Mice were maintained on a 12:12 h light–dark cycle with lights on at 07:00 h and allowed access to food and water ad libitum. All mice were acquired and cared for in accordance with the guidelines published in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publications No. 85–23, Revised 1985) and adhered to the National Institutes of Health and the American Association for Laboratory Animal Science guidelines. The protocols used in this study complied with the regulations of the Institutional Animal Care and Use Committee at the Northeastern University and adhered to the ARRIVE guidelines for reporting in vivo experiments in animal research [23].

Drug preparation and administration

CBD was a gift from the Center for Drug Discovery (Northeastern University, Boston MA) and dissolved in EtOH/cremophor/saline 1:1:18 for I.P. injections. Following acclimation, mice were randomly assigned to one of four groups corresponding to EtOH/cremophor/saline vehicle, 3, 10, or 30 mg/kg I.P. CBD. The amount of drug was adjusted to deliver vehicle and each dose in a volume of 0.2 ml. To deliver drug remotely during the imaging session, a poly-ethylene tube (PE-20), approximately 30 cm in length, was positioned in the peritoneal cavity. The range of doses of CBD evaluated were taken from the literature [24–26].

Awake mouse imaging

Imaging system

We used previously described awake mouse imaging techniques [27]. Briefly, we used a quadrature transmit/receive volume coil customized for optimal space filling, anatomical resolution, and signal-to-noise. The mouse holder (Ekam Imaging; Boston, MA) fully stabilizes the head in a cushioned helmet, minimizing discomfort caused by ear bars and other restraint systems that are commonly used to immobilize the head for awake animal imaging. A movie showing the set-up of a mouse for awake imaging is available at http://www.youtube.com/watch?v=W5Jup13isqw. The effectiveness of this passive restraining system can be judged by the minimal level of motion artifact recorded during the imaging session as shown in Additional file 1: Figure S1. The average displacement in any orthogonal direction over the entire 15 min scanning session did not exceed 56 µm.

Acclimation

A week before imaging, mice were acclimated to the head restraint and the noise of the scanner [27]. The acclimation protocol was repeated over four consecutive days reducing autonomic nervous system-induced effects during awake animal imaging (e.g., changes in heart rate, respiration, corticosteroid levels and motor movements), to improve contrast-to-noise ratios and image quality [28]. Only mice that habituate to restraint were used in the analysis. Additionally, three mice died and five were lost to motion artifact or technical complications resulting in group sizes of EtOH/cremophor/saline vehicle (n = 8), 3 mg/kg (n = 6), 10 mg/kg (n = 5), and 30 mg/kg (n = 7).

BOLD phMRI and pulse sequence

Experiments were conducted using a Bruker Biospec 7.0T/20-cm USR horizontal magnet (Bruker; Billerica, MA) and a 20-G/cm magnetic field gradient insert (ID = 12 cm) capable of a 120-µsec rise time. At the beginning of each imaging session, a high-resolution anatomical data set was collected using the rapid acquisition relaxation enhanced (RARE) pulse sequence (18 slices; 0.75 mm; field of view (FOV) 1.8 cm; data matrix 128 × 128; time to repeat (TR) 2.1 s; time to echo (TE) 12.4 ms; Effective TE 48 ms; number of averages (NEX) 6; 6.5 min acquisition time). Functional images were acquired using a multi-slice H alf Fourier A cquisition S ingle Shot T urbo Spin E cho (HASTE) pulse sequence (18 slices; 0.75 mm; FOV 1.8 cm; data matrix 96 × 96; TR 6 s; TE 4 ms; Effect ET 24 ms; 15 min acquisition time; in-plane resolution 187.5 µm 2 ). Spin echo is required to achieve the high anatomical fidelity required for data registration to the mouse MRI atlas as shown in Additional file 1: Figure S2 [29]. Each functional imaging session consisted of uninterrupted data whole brain scans, 150 scan repetitions, total elapsed time 15 min. The control window included the first 50 scan repetitions, a 5 min baseline. Following the control window, an I.P. injection of drug was given followed by a 10 min stimulation window consisting of acquisitions 50–150.

The dose-dependent effect of CBD on brain activity was quantified by measuring positive and negative percent changes in BOLD signal relative to baseline as previously described [30]. A complete description of the data analysis is provided in Additional file 1: phMRI analysis.

Resting state functional connectivity

Sixty min prior to imaging mice were injected I.P. with EtOH/cremophor/saline vehicle (n = 10) or 10 mg/kg CBD (n = 10). The mice were then anesthetized and fitted into the coil as described above. Mice were maintained under light 1% isoflurane anesthesia (ambient air mix), adjusted to hold the respiratory rate between 50–60 breaths/min as compared to a normal rate of 85–90 breaths/min. Scans were collected using a spin-echo triple-shot EPI sequence (imaging parameters: matrix size = 96 × 96 × 20 (H × W × D), TR/TE = 1000/15 ms, voxel size = 0.312 × 0.312, slice thickness = 1.2 mm, with 200 repetitions, total time 10 min. The data processing, normalization and group level analysis is described in detail in Additional file 1.

Resting state BOLD functional connectivity analysis

Degree centrality

All network analysis was computed with Gephi, an open-source network analysis and visualization software [31]. Absolute values of the CBD and vehicle symmetric connectivity matrices were imported, and edges were loaded as undirected networks. A complete description of the graph theory analysis is provided in Additional file 1: Graph Theory Analysis.

CBD analysis

To validate that there was a dose-dependent change in CBD levels, serum levels of cannabinoids were analyzed [22]. In brief, methanolic extracts of 90 µl of serum were partially purified on C18 solid phase extraction columns (Zorbax) and eluants were analyzed using HPLC/MS/MS (API 3000, Applied Biosystems). Deuterium-labeled anandamide elutes in the same fraction as CBD and was used as an internal standard to monitor recovery. Levels of CBD and THC were analyzed using standard curves with Analyst Software as previously described [22]. During analysis it was discovered that each of the samples contained a small fraction THC in addition to CBD. This can occur during synthesis and is often unknown if the levels are not analyzed. The plasma ratio in each dose was ca 25:1 CBD:THC (Additional file 1: Figure S3B).

Results

Table 1

Positive bold volume of activation

This table is a truncated list of 35 out of 138 brain areas ranked in order of their significance for change in positive BOLD volume of activation (number of voxels). Reported is the median number of voxels significantly activated 10 min post injection of vehicle (0), 3, 10 and 30 mg/kg I.P. doses of CBD. Show are p values and effect size (omega square ω 2 ) for each brain area. The significance levels for FDR was p ≤ 0.051. Areas highlighted in gold are associated with the olfactory system and prefrontal cortex. Areas highlighted in green are in the hindbrain brainstem and cerebellum. The areas highlighted in blue are located between the forebrain and hindbrain areas

Polarized Positive and Negative BOLD. The color-coded 3D reconstructions for positive and negative BOLD denote the location of the brain areas comprising the hindbrain, midbrain, and forebrain, respectively. The bar graphs below show the average median number of voxels from each of these brain regions for vehicle, 3, 10, and 30 mg/kg I.P. doses of CBD. For forebrain positive BOLD: (****p < 0.0001, 10 mg >Veh); (*p = 0.0133, 10 mg > 3 mg). For hindbrain positive BOLD: (**p = 0.0012, 10 mg < Veh); (****p < 0.00001, 10 mg < 3 mg); (**p = 0.0019, 10 mg < 3 mg). For forebrain negative BOLD: (*p = 0.0197, 10 mg < Veh); (**p = 0.0043 10 < 3 mg). For midbrain negative BOLD: (*p = 0.0389; 3 mg >Veh); (***p = 0.0006, 3 mg > 30 mg). For hindbrain negative BOLD:D voxels (median ca 16) that is significantly increased with the 10 mg dose of CBD over vehicle and 3 mg (****p ≤ 0.0001, 10 mg > Veh, 10 mg > 3 mg)

Shown in Table ​ Table2 2 is a truncated list of 50 out of 138 brain areas ranked in order of their significance for change in negative BOLD volume of activation. A false discovery rate for multi-comparisons gives a significance level of p ≤ 0.073. The areas highlighted in gold are again the olfactory bulb and prefrontal cortex, as in Table ​ Table1, 1 , but the pattern is reversed, with areas like the glomerular layer and orbital cortex showing a U-shaped dose response. The 3 mg/kg dose is most effective in causing a negative change in BOLD signal while the 10 mg/kg dose is least effective. This reversed pattern between positive and negative BOLD is also true for the areas highlighted in green representing the hindbrain, brainstem, and cerebellum. The 10 mg/kg dose is most effective in causing a negative change in BOLD signal with many areas presenting with the inverted U-shaped dose response. The brain areas highlighted in blue are located along the rostral/caudal axis between the forebrain and hindbrain (Fig. 1 ).

Table 2

Negative BOLD volume of activation

This Table is a truncated list of 50 out of 138 brain areas ranked in order of their significance for change in negative BOLD volume of activation. Reported is the median number of voxels significantly activated 10 min post injection of vehicle (0), 3, 10 and 30 mg/kg I.P. doses of CBD. Show are p values and effect size (omega square ω 2 ) for each brain area. The significance level for FDR was p ≤ 0.073. The areas highlighted in gold are the olfactory bulb and prefrontal cortex. Areas highlighted in green are localized to the hindbrain brainstem, and cerebellum. The brain areas highlighted in blue are located along the rostral/caudal axis between the forebrain and hindbrain

This relationship between CBD doses and positive BOLD was reversed for negative BOLD (Fig. 1 b). Unlike positive BOLD areas highlighted in gold, olfactory bulb, and prefrontal cortex, show a U-shaped dose response. The 3 mg/kg the most effective in causing a negative change in BOLD signal while 10 mg/kg was least effective. This reversed pattern between positive and negative BOLD is also true with the hindbrain brainstem, and cerebellum showing an inverted U-shape, with 10 mg/kg dose stimulating the strongest negative BOLD signal (Fig. 1 b). In the forebrain, there is a baseline of negative BOLD voxels (median ca 40) that is significantly reduced with either 10 mg/kg (p = 0.0197) or 3 mg/kg CBD (p = 0.0043) versus vehicle. In the hindbrain, the baseline of negative BOLD voxels (median ca 16) was increased with both 10 mg/kg and 3 mg/kg CBD (p ≤ 0.0001 for each comparison) over vehicle. In the midbrain brain areas, 3 mg/kg increased the median number of negative voxels over vehicle (p = 0.0389), whereas 30 mg/kg was associated with a lower number of negative voxels than the 3 mg/kg (p = 0.0006).

Figure 2 summarizes the effect of the 10 mg/kg of CBD on BOLD signal, with tables showing significant changes, negative and positive, BOLD activation. The 3D image summarizes the location of the brain areas presenting with positive (red) and negative (blue) activation. The distribution is polarized along the rostral-caudal axis with positive BOLD localized to the forebrain and negative signal changes confined to the hindbrain. The forebrain areas are represented by the olfactory system (e.g., granular and glomerular layers of olfactory bulb, anterior olfactory area, and tenia tecta) and the prefrontal cortex (e.g., prelimbic, frontal association, orbital, infralimbic, 2nd and primary cortices). Connecting these bilateral forebrain areas is the forceps minor of the corpus callosum. The negative BOLD in the hindbrain is represented by the cerebellum (e.g., 2nd–6th lobules, simple lobule, crus of ansiform lobule flocculus) and ascending reticular activating system (ARAS) (e.g., dorsal raphe, parabrachial nucleus (n.), parvicellular reticular n., gigantocellularis, pedunculopontine n., pontine reticular n. mesencephalic reticular n.). In addition to volume of activation i.e. number of voxels activated with CBD treatment, changes in positive and negative BOLD signal over time, another measure of functional activity, are presented for the ARAS and forebrain. Each time point or image acquisition is the average BOLD signal of all brain areas comprising the ARAS and all areas in the forebrain. CBD has no significant effect on positive BOLD signal in the ARAS; instead, vehicle causes a greater, albeit small and at the level of threshold (above noise) increase in CBD (2-way ANOVA, F(1,88) = 8.48, p = 0.0045; CBD < Veh). In the forebrain this pattern was reversed. CBD caused a significant increase in positive BOLD versus vehicle (F(1,167) = 6.025, p = 0.0199; CBD > Veh) while being significantly less than vehicle for negative BOLD (F(1,167) = 5.008, p = 0.026 CBD < Veh).

Acute Effects of 10 mg Dose of CBD. The tables show a truncated list of 31 and 13 out of 138 brain areas ranked in order of their significance for changes in negative and positive BOLD volume of activation, respectively in response to the 10 mg/kg I.P. dose of CBD. The 3D image (a) summarizes the location of these brain areas presenting with positive (red) and negative (blue) BOLD volume of activation. Below are graphs of BOLD signal change over the 15 min imaging session for the ascending reticular activating system (ARAS) (b) and forebrain (c). Comparisons are made between vehicle and the 10 mg dose of CBD. Shades of red denote positive changes and shades of blue negative changes. For ARAS positive BOLD: (p = 0.0045; CBD < Veh). For ARAS negative BOLD: (p < 0.0001; CBD >Veh). For forebrain positive BOLD (p = 0.0199; CBD > Veh). For forebrain negative BOLD: (p = 0.026 CBD > Veh)

Looking at changes in connectivity (degrees) between CBD and controls in major brain areas reveals a pattern of functional coupling consistent with the pronounced negative BOLD observed with phMRI (Fig. 3 ). Mice treated with 10 mg/kg CBD prior to imaging showed a significant decrease in coupling in all the hindbrain regions, midbrain, hypothalamus, and cortex. While there were no significant differences seen in forebrain regions of olfactory system, prefrontal cortex, the thalamus, or the amygdala. Interestingly, when the ARAS is combined as a node, the same pattern of uncoupling appears between the ARAS and all these major brain regions, except for the olfactory system, prefrontal cortex, thalamus, and amygdala (Additional file 1: Figure S4). The ARAS as a node, under the influence of CBD, may not be significantly less than these brain regions but it is positively correlated with specific brain areas within these areas (Fig. 4 ). The 2D maps show the neuroanatomical position of brain areas with increased coupling to the ARAS (highlighted in red) following CBD treatment compared to vehicle. The areas shown in gray comprise the ARAS. A 3D reconstruction of these areas is shown to the left. The olfactory system is a large part of the hyperconnectivity between the ARAS and these limbic forebrain regions. The wire diagram below shows the significant negative (blue) and positive (red) connections between the ARAS and specific areas in the primary olfactory system.

Regional Changes in Connectivity. Shown are 3D color coded images summarizing CBD-induced changes in connectivity (a) and box and whiskers plots (b) depicting differences in degree centrality in various subregions between vehicle and rats treated with 10 mg/kg CBD 60 min prior to imaging. The CBD group had significantly lower degree centrality of nodes within the hippocampus, hypothalamus, cortex, cerebellum, brainstem, basal ganglia, midbrain, and pons (**p < 0.05, not significant = ns). There were no significant differences in degree within nodes of the amygdala, olfactory system, prefrontal cortex, or thalamus

Hyperconnectivity to the ARAS with CBD Treatment. Shown to the left (a) are 2D axial maps showing the location of brain areas (red) with enhanced coupling to the ARAS following CBD treatment. Areas in gray denote the location of brain areas comprising the ARAS. The 2D images are summarized in the 3D reconstruction of the red and gray brain areas (b). The circle of connections beneath (c), display the neighboring nodes of the ARAS in the CBD treated group within the olfactory system. Nodes that have a greater degree centrality in the CBD group have been colored red, while nodes that have a greater degree centrality in the vehicle group have been colored blue. Node size has been scaled to reflect the relative difference in degree centrality between the vehicle and CBD group, with the larger nodes reflecting a larger difference in degree centrality

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Shown in Fig. 5 are autoradiograms of in situ hybridization of NAPE-PLD from the Allen Brain Atlas [32]. These are sagittal sections that extend from the midline laterally from top (A.) to bottom (B.). The signal intensity reflects the level of mRNA in specific brain areas. Note, in general, the forebrain olfactory system and hindbrain cerebellum have a high density of NAPE-PLD mRNA. All the areas comprising the ARAS have NAPE-PLD mRNA (A. gigantocellularis, pontine reticular n, oral, midbrain reticular n., pontine reticular n. caudal; B. parabrachial n., pedunculopontine n., parvicellular reticular n.). Many of the areas shown in the 2D maps of positive ARAS connectivity to thalamus, amygdala, prefrontal, and olfactory system show high levels of NAPE-PLD mRNA (e.g., olfactory bulb, anterior olfactory n., tenia tecta, infralimbic ctx, lateral preoptic area, ventral medial hypothalamus, central amygdala, geniculate, reticular n. pretectal n.).

N-acyl-phosphatidylethanolamines -specific phospholipase D mRNA. Shown are autoradiograms of in situ hybridization of NAPD-PLD messenger RNA in mouse brain. The sagittal section A and B extend medial to lateral. Abbreviations PAG—periaqueductal gray. Image credit: Allen Institute

Discussion

CBD produced activation in the prefrontal cortex and deactivation in the brainstem/cerebellum, particularly within the ascending reticular activating system (ARAS), with no changes apparent in the hypothalamus, amygdala, basal ganglia, or hippocampus. rsFC showed a decoupling of the hindbrain and midbrain regions, particularly the ARAS following CBD treatment. The integrated activity of the ARAS affects all aspects of cognitive and emotional behavior [33]. Interestingly, there were several areas of the brain that were positively coupled with the ARAS following CBD treatment. These area colocalize with a high density of N-acyl-phosphatidylethanolamines (NAPE) by a NAPE-specific phospholipase D (NAPE-PLD) mRNA [32]. NAPE-PLD is a constitutively active enzyme involved in the biosynthesis of N-acylethanolamines, signaling lipids molecules like anandamide [34]. The rsFC showed hyperconnectivity and hypoconnectivity that was consistent with the phMRI data. These findings are discussed with respect to the many studies showing CBD can affect the emotional and cognitive behavior associated with anxious and fearful events and NAPE-PLD as a putative mechanism of action.

Human imaging and CBD

Several studies have used imaging to characterize the acute effect of CBD on brain activity in humans. SPECT imaging in volunteers diagnosed with anxiety disorder shows CBD increases blood flow in the cingulate cortex and reduces flow in the hippocampus while decreasing anxiety [35]. BOLD imaging in healthy volunteers, shows CBD decreases activation in the cerebellum, anterior cingulate, and amygdala, in a visual fear paradigm but not to neutral stimuli [15]. In healthy volunteers, CBD enhances caudate and hippocampal activation and fronto-striatal connectivity during salience processing [12, 13] and under resting state conditions [16], enhances auditory and visual processing [19] and effects working and episodic memory associated with an increase in blood flow to the hippocampus [14]. CBD alters functional coupling in cerebellum, frontal, and occipital cortices in patients with treatment resistant epilepsy [17] and attenuates hippocampal-striatal functional connectivity in psychosis patients [18]. All these studies gave oral doses of CBD prior to scanning, thus establishing a baseline of resting state blood flow that changed with different task-related paradigms or differed from placebo or healthy controls in response to a preexisting condition.

Polarized positive and negative BOLD

The data reported here in awake mice are not easily compared to the human imaging studies. As the only study of its kind, we are looking at the immediate, dose-dependent effects of CBD, administered I.P., on brain activity across 138 different brain areas. The dose response showed the same inverted U-shape reported in many behavioral studies following systemic injection of CBD in rodents [24, 25, 36, 37] and humans [38]. The 10 mg/kg I.P. dose stood out as being the most effective, corroborating the many studies in rodents employing this dose [24–26, 36]. Within 10 min of injection, there was increase in positive BOLD signal in the prefrontal cortex/olfactory system and negative BOLD signal in the brainstem/cerebellum, particularly in brains areas comprising the ARAS. The absence of BOLD signal change in brain areas between the rostral/caudal axis of the brain (e.g., hippocampus, sensorimotor cortices, thalamus, hypothalamus, amygdala, and basal ganglia) made this pattern of activation and deactivation especially intriguing. This is unlike anything reported in awake animal imaging following tests on numerous CNS active drugs [8–11, 39–47]. Here we show the positive and negative changes in BOLD signal occur within 10 min of injection, and while CBD is known to rapidly penetrate the brain within seconds following systemic administration [48], its effects could be orchestrated easily by both peripheral and central targets. It should be noted that a pharmacokinetics study by Holzek et al. reported a much slower time course for brain penetrance following systemic CBD treatment [49].

CBD targets

The primary targets for systemic CBD are unknown. Possible candidates include the cannabinoid CB1 receptors [50, 51], serotonin 5HT1a receptor, and the transient receptor potential vanilloid type 1 (TRPV1) [7, 52]. One important consequence of systemic CBD is the dramatic change in the CNS lipidome including increases in anandamide and related lipids that occurred in a NAPE-PLD dependent manner [22]. Do the CBD induced site-specific change in brain activity reported in our study match the distribution the putative targets noted above? CB1 receptors are localized to olfactory system, hippocampus, basal ganglia, cerebellum, and neocortex but very little in brainstem [53]. High densities of 5HT1a receptors are localized to prefrontal cortex, amygdala, hippocampus, and hypothalamus, while receptors are undetectable in the cerebellum and marginal in the brainstem [54, 55]. TRPV1 is expressed throughout the CNS with the highest density of receptors localized to the hippocampus, amygdala, hypothalamus, prefrontal cortex, and cerebellar cortex, while the lowest levels are in the brainstem [56–58]. NAPE-PLD distribution as shown in Fig. 5 is highest in hippocampus, cerebellum, olfactory system, and site-specific areas of the thalamus, amygdala, and brainstem [59, 60]. The distribution of NAPE-PLD seems to fit the activity pattern of CBD, specifically with respect to the ARAS. However, neither the distribution of CB1, 5HT1a, TRPV1, nor NAPE-PLD alone or together, can explain the absence of responsiveness of large parts of the brain to CBD or the polarization of BOLD signal. CBD has a complex pharmacology with activity at multiple targets beyond those discussed above (review see [7]). Given the promiscuity of CBD, there is no obvious explanation for the pattern of BOLD signal change based on location of a single target in the brain.

Autonomic arousal and stress

The negative BOLD in brain areas that comprise the ARAS would suggest a decrease in brain activity and a reduction in autonomic arousal. Acute and chronic dosing of CBD in humans and animals has no appreciable effect on blood pressure, heart rate or blood flow [61]. However, CBD mediates the emotional and cardiovascular response to stress. CBD blunts the increased heart rate and blood pressure associated with the stress of forced immobilization [26, 62, 63] and increase in blood pressure, heart rate and immobility behavior in response to fear associated with the memory of an aversive condition [64]. CBD reduces immobility and escape behavior in mice exposed to a wild snake [65], altering the innate fear and aversion to predation. Rats exposed to cats present with long-lasting anxiogenic behavior that can be reduced with CBD [66]. Thus, CBD can reduce the anxiety, fear and immobilization associated with stressful or life-threatening events.

Speculation

Interesting by its very nature, awake fMRI is a model of restraint stress, requiring the immobilization of the head to minimize artifacts. Acclimation is used to reduce the autonomic measures of stress [28], meaning the test subjects have a history of stress and adaptation. In additional, there is probably some emotional/physical stress associated with drug delivery during testing. The deactivation of the ARAS as interpreted by the increase in negative BOLD would be anticipated under these conditions and provide a neural target for CBD that would explain the reduction in autonomic and behavioral responses associated with anxious and potentially harmful environmental stimuli. Is the negative BOLD response to the ARAS unique to the acclimation process, i.e., is it an adaptation that has primed the lipidome to function under a new set of environmental pressures?

Evolutionary significance

Is there a neurobiological explanation in the evolution of animals that would favor the global pattern of deactivation and uncoupling of functional circuits observed in much of the brain while favoring activation and hyperconnectivity to the forebrain and olfactory system by the ARAS? CDB is most effective when given to patients or animals presenting with high anxiety and fear. Specifically, it can reduce heart rate and blood pressure during heighten sympathetic arousal but has little to no intrinsic effect on these autonomic measures under homeostatic conditions. CBD is acting on reactive neural circuitry—the brain’s prewired, immediate response to threat. Freezing or behavioral arrest is a natural response to predator threat as a way of reducing detection. However, when the interaction becomes physical, the behavioral arrest can escalate into tonic immobility, an innate response of extreme physical inactivity [67]. This last chance to escape predation is commonly referred to as death feigning [68]. The immobility arises from descending neurons in the medullary, pontine reticular formation that suppress spinal motor neuron activity [69]. The neural circuitry of tonic immobility includes much of the ARAS described here [70–72], in addition to the PAG [73, 74], basolateral and central amygdala [75], and medial dorsal thalamus [76]. Treatment with CBD affects the BOLD signal and rsFC in all these areas. One of the more fascinating aspects of tonic immobility is continued sensory perception, i.e., animals feigning death can process sensory information and are aware of their environment [72, 77, 78]. The activation of the olfactory system by CBD would allow animals to continually survey their environment for the presence of the predator. The hypothesis that CBD could be affecting endocannabinoid signaling through NAPE-PLD is purely speculative but given the unique pattern of global brain activity caused by CBD treatment may warrant investigation and has far reaching implications. The neuropsychiatric trauma associated with life threatening experiences, e.g. PTSD, may crystalize around this phylogenetically old neural circuitry primal to survival [79]. Indeed, the evidence for CBD and endocannabinoid signaling playing a significant role in emotional regulation in neuropsychiatric disorders is growing [80].

Limitations

(1) These studies did not address sex difference in CBD responsivity. The previous study investigating CBD’s effects on the brain lipidome were in female mice [22], so there is evidence that CBD has a significant effect on the brain within the time period; however, future studies will need to address if the changes in brain connectivity shown here in male mice are also measured in female mice. (2) Resting state functional connectivity was collected while rat were lightly anesthetized with isoflurane to minimize motion and physiological stress during “resting state” BOLD functional connectivity imaging (review see [81]). Anesthesia may reduce the magnitude of the BOLD signal but does not disrupt the connectivity as demonstrated across species and under different physiological conditions [82–86]. In this study the rsFC showed hyperconnectivity and hypoconnectivity that was consistent with the phMRI data. (3) There were no measures of NAPE-PLD activity in response to CBD challenge. These studies were not originally designed to test the involvement of NAPE-PLD.

Summary

phMRI in awake mice was used to assess the immediate dose-dependent effects of CBD on global brain activity. The pattern of brain activity was unique and unexpected, characterized by activation in the prefrontal cortex and deactivation in the brainstem/cerebellum, particularly in the ARAS. These data provide a novel framework to understand how CBD drives CNS changes that can be targeted for therapeutics. The putative target and mechanism of action is NAPE-PLD the enzyme responsible for the biosynthesis of lipid signaling molecules like anandamide.

Can CBD Oil Treat Horses With Cushing’s?

Equine Cushing’s disease, medically known as pituitary pars intermedia dysfunctions (PPID), is an endocrine (hormone) disease affecting the pituitary gland in horses.

The pituitary gland is located at the base of the brain, and it’s the master gland responsible for hormone production for brain signaling which affects the whole body. It can be a devastating diagnosis for horse owners. The disease primarily affects horses over the age of 10, with 19 years being the average age of diagnosis [1].

As more research about the benefits of CBD oil emerges for humans and animals, vets and equestrians alike are turning to CBD to support their horses’ health.

In this article, we’ll take an in-depth look at what the current research says about CBD and this metabolic syndrome.

Symptoms Of Cushing’s Syndrome In Horses

Pituitary pars intermedia dysfunctions (PPID) or Cushing’s disease is an example of metabolic syndrome that increases the risk of heart disease and strokes. It’s a very slow and progressive disease, so early veterinary intervention can help to improve your horses’ quality of life.

In PPID, the pituitary gland—also known as the master hormone gland—becomes overactive and produces large amounts of the adrenocorticotropin hormone (ACTH). Not much is known about what causes PPID, but it has been observed to occur from a benign pituitary tumor most prevalent in older horses.

One of the functions of ACTH is to stimulate the release of the stress hormone, cortisol. When left untreated, elevated ACTH increases cortisol levels in your horses’ body that can lead to very serious consequences.

Clinical signs that a horse may have Cushing’s syndrome include:

  • A pot-bellied appearance
  • Drastic changes in weight
  • Abnormal fat distribution, especially in the face
  • Lethargy
  • Muscle loss
  • Increased coat length, and failure to shed coat in summer
  • Slow wound healing
  • Prone to infections
  • Excessive drinking and urinating
  • Development of laminitis

If you notice these changes in your horse’s body and see a noticeable difference in its mood, you should seek professional advice from your vet to get a proper diagnosis.

Cushing’s is a very slow and progressive disease, so early veterinary intervention can help to improve your horses’ quality of life.

Diagnosing Cushing’s In Horses

The most common way to test for Cushing’s is with the low-dose dexamethasone suppression (LLD) test.

This test is typically conducted overnight and requires a baseline blood sample for cortisol levels. The vet will then administer a dose of dexamethasone and test blood samples 18–20 hours later. In a healthy horse, the cortisol levels will decrease from the injection of dexamethasone. However, horses with Cushing’s will still have elevated cortisol levels.

Insulin resistance is common in equines diagnosed with Cushing’s so a blood glucose or insulin test is also recommended in conjunction with testing for Cushing’s.

What Are Conventional Treatments For Cushing’s Disease In Horses

A Cushing’s diagnosis is devastating. While there is no cure for PPID, with veterinary treatment, a balanced nutrition plan, and careful management, many horses and ponies can live comfortable and active lives for many years after their diagnosis.

Most vets will recommend a focus on a low-carb diet to maintain healthy blood sugar levels and weight in your horse and careful monitoring every six to eight months to track the progression of PPID.

When it comes to medications, your vet may prescribe dopamine, serotonin, and cortisol agonists. The most common medication for PPID is dopamine agonists, which help to control the overactive pituitary gland.

Some of the negative side-effects of dopamine agonist medications include:

  • Colic
  • Digestive issues
  • Depression
  • Weight loss

What Is CBD?

CBD or cannabidiol is a naturally derived compound from cannabis plants. It’s one of over a hundred compounds in a class called cannabinoids.

These cannabinoids look very similar to neurotransmitters in mammal bodies called endogenous cannabinoids in a system called the endocannabinoid system (ECS). The ECS is responsible for maintaining homeostasis (balance) in a wide variety of functions, including the sleep-wake cycle, stress, metabolism, mood, and the immune system.

CBD is not to be confused with its more notorious cousin, THC (tetrahydrocannabinol). Unlike THC, CBD does not produce intoxicating effects, which makes it a more reliable compound in terms of supporting one’s health with daily use.

Hemp crops have high concentrations of CBD, and that’s where legal CBD comes from. Thanks to the US Farm Bill in 2018, hemp crops that contain up to 0.3% THC were legalized, while marijuana plants (anything with over 0.3% THC) remain federally illegal.

CBD is incredibly versatile and can be found in anything from capsules, sublingual oils, topical treatments, and even pet treats and food.

How Does CBD Work For Horses?

The endocannabinoid system (ECS) is a major signaling system that exists in humans and our furry pets. There’s still limited research surrounding horses for cannabis-related studies. However, researchers agree that CBD appears to stimulate horse’s ECS in the same way it does in humans.

CBD, even in high doses, is well-tolerated and can provide positive uses to support health and wellness with very few adverse effects.

Cannabinoids such as CBD, CBG, CBN, and THC from hemp and naturally produced cannabinoids in the body (endogenous) communicate in the intricate endocannabinoid system’s receptors to turn on key functions to maintain homeostasis (balance) for optimal body function.

When we become out of balance in one system for too long, such as poor stress levels, irregular sleep cycles, or disrupted hormone function, horses and humans become prone to serious health issues. Supplementing your equine’s diet with CBD allows the endocannabinoid system to function more effectively to help deal with stress better for improved wellness.

CBD For Horses And Its Profound Benefits

Support ECS For Healthy Inflammatory Response

One of the roles of the endocannabinoid system is to regulate healthy levels of inflammation.

CBD products help to increase the body’s levels of endocannabinoids and slows the breakdown of these neurotransmitters to improve the ECS function.

Horses with a Cushing’s diagnosis are susceptible to insulin resistance, obesity, and cardiovascular disease, which are all linked with inflammation.

The CB2 receptor plays an important role in regulating healthy inflammation. CBD may help to improve the levels of endogenous cannabinoids for improved communication with CB2 receptors for gentle and natural-based support in the immune system [2].

Support ECS For Comfort Levels In Your Horse

Equines with a Cushing’s diagnosis are more susceptible to laminitis, a condition that affects the tissues binding the hoof wall to the pedal bone. Since PPID affects hormone function and can result in excessive weight gain, it can put a lot of pressure on these joints causing tremendous pain.

CBD oil may help to support comfort levels in your horse. As we mentioned in the previous point, CBD helps the ECS regulate inflammation. While inflammation is a healthy response to support wound healing and fight off infection, too much inflammation can result in pain and tissue damage as seen in laminitis.

Support Healthy Recovery Post-Exercise

For horses that undergo a lot of training, CBD may help to support your equine’s muscle recovery, to keep them in their healthiest form. Adding CBD oil to your horses’ wellness regime may help with their performance as it may help to support post-exercise recovery and maintain healthy levels of stress.

Calm Your Horse

CBD has been studied for its calming and relaxing benefits, which may help to combat the negative effects of stress.

Stress is an important protective mechanism for the fight-or-flight response, but it was never meant to be turned on 24/7. Chronic stress leads to a wide host of health conditions that presents themselves in Cushing’s syndrome in horses.

Many trainers reach for CBD oil to keep their horses calm in stressful situations like transportation or when adapting them to a new environment.

Frequently Asked Questions About Horses & CBD

How Much CBD Should I Give My Horse?

Dosing CBD for humans and animals is always a tricky subject as it depends on many different factors and can take some experimentation to find the ideal dose. CBD is typically dosed depending on weight, so you’ll need much more CBD for your horse than you would for yourself or your dog.

Look for CBD products marketed for horses, as they will have more appropriate strengths suited to the size of your horse, and follow the instructions with the lowest dose and monitor your horses’ behavior before increasing the dose.

If you plan on giving CBD products to your horse to support a health condition, we strongly advise that you speak with your veterinarian to ensure that it’s the right thing to do. We want to underscore that there is no evidence that CBD can treat horses with Cushing’s.

How Do I Give My Horse CBD?

Edible CBD oil and treats are the most common products marketed for horses, one of the easiest ways to give your horse CBD is to disguise it in their feed or to give it to them straight in their mouths if they’re not fussy.

At Neurogan, we offer CBD hemp pellets for horses. These palettes are made with one simple ingredient, high-quality, sun-grown hemp. They provide the benefits of the cannabinoids and terpenes from hemp and are an excellent source of fiber and antioxidants that may help reduce oxidative stress.

Will CBD Show Up On A Drug Test For Competition Horses?

CBD and other metabolites from hemp-based cannabinoids may be detected in blood and urine samples for up to 2 weeks after taking it and may result in a failed drug test.

When it comes to racing horses, this may disqualify them from competing, but more ongoing research is underway to clarify the rules surrounding legal hemp extractions and horse racing as more benefits emerge supporting CBD for equine health.

What Are The Side-Effects Of CBD In Horses?

CBD has been shown in multiple studies to be a well-tolerated compound even at high doses, so it’s relatively safe [3]. That being said, it’s not completely without potential side effects.

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Luckily, the adverse effects of CBD wear off when it leaves your system, and it’s more likely to happen when you give your horse too much CBD.

Some of the side-effects of CBD to look out for in your horses include:

  • Drowsiness
  • Diarrhea
  • Dry mouth

Our pets can’t let us know when something’s wrong, so always monitor your horse after giving them CBD to ensure there are no negative interactions and to mitigate the chances of inducing a negative experience.

How To Buy CBD For Horses

When it comes to shopping for pet products in the CBD space, it can be even trickier to decipher the legitimate products from snake oil.

The food packaged for animals typically comes from off-cuts and isn’t as high of quality or nutrient-dense as food for humans. When you love animals as much as we do, we make sure our animals are getting the best quality possible when it comes to food and CBD products.

Here’s what you can do to make sure you’re buying the highest-quality CBD products for your horses.

1. Know Where The Hemp Was Grown

Look to make sure the source of hemp was grown using the best farming practices. High-quality CBD oil starts with a clean hemp source. Unfortunately, not all brands take hemp sourcing seriously. While the United States is known for its high agricultural standards, the same can’t be said for other countries’ hemp crops.

Hemp is highly sensitive to its growing environment. It can absorb many contaminants such as heavy metals and pesticides in the soil and surrounding area, which is why farming practices and hemp sourcing is critical when shopping for CBD products to support your animal’s health.

2. Buy Full Spectrum Or THC-Free Broad Spectrum Extracts

There is more than one beneficial compound found in hemp. In fact, there are over a hundred cannabinoids and dozens of terpenes in hemp that can support the effects of CBD in the endocannabinoid system.

To leverage the full benefits that hemp has to offer, you want to give your horses a CBD product that works as hard as they do. Full spectrum extracts are the least processed and more natural form of hemp extracts, they contain as much of the natural plant profile, which means it can include up to 03% THC.

If you’re looking for a THC-free option, broad spectrum CBD is your best bet. It undergoes further processing to filter out THC, while still providing whole-plant synergy for a more potent CBD product.

CBD isolate or distillate products only contain one active ingredient from hemp—CBD. It also tends to be cheaper as it requires less careful extraction methods from the cannabis plant, and manufacturers can sell it in bulk at cheaper prices. CBD isolate on its own can still provide holistic wellness benefits, but it won’t be as potent and increase the risk of inducing adverse effects.

3. High Potency CBD

CBD oil is typically dosed based on size. Since horses way much more than humans do, make sure the CBD products you’re buying have the appropriate strength to support a horse’s endocannabinoid system. Shop with brands that have a line of CBD products for horses as the potency tends to be much higher (upwards of 4000 MG/1 oz bottle).

The Takeaway: Can CBD Oil Help With Cushing’s?

There is no cure for Cushing’s syndrome in horses, but CBD may help improve your equine’s quality of life. With veterinary care and changes in nutrition, horses can enjoy a comfortable life for years to come after a diagnosis.

CBD does show a lot of promise for supporting a healthier lifestyle in humans and animals, but we always recommend you speak with your veterinarian before giving CBD to your horse with Cushing’s.

If you’re looking for more articles like this on how to give your pet a happier life with CBD, be sure to check out our other blog posts or sign up for our newsletter to receive the Inside Scoop on the latest findings in the CBD industry straight to your inbox.

Marijuana Use and Hypothalamic-Pituitary-Adrenal Axis Functioning in Humans

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Abstract

Preclinical studies suggest cannabinoids affect functioning of the hypothalamic-pituitary-adrenal (HPA) axis, but little is known about the effects of marijuana (MJ) use on HPA axis functioning in humans. Since previous work indicates substances of abuse may dysregulate the HPA axis, it is critical to understand how MJ use affects HPA axis activity. Here, we review studies that (a) examined the effects of acute MJ administration on HPA axis functioning, (b) investigated the impact of stress on HPA axis functioning in MJ users, (c) examined the effect of chronic MJ use on basal cortisol levels, and (d) studied the relationship between MJ use and the cortisol awakening response (CAR). Findings indicate acute MJ administration typically raises cortisol levels, but this increase is blunted in MJ-dependent users relative to controls. Frequent MJ users have blunted adrenocorticotropic hormone and cortisol reactivity in response to acute stress. These findings suggest HPA axis activity may be dysregulated by heavy MJ use. Alternatively, dysregulation of the HPA axis may be a risk marker for heavy MJ use. There is mixed evidence for how MJ use affects basal cortisol levels and the CAR. Future studies should consider MJ use characteristics, method of hormone collection, time when samples are collected, and environmental factors that may influence HPA axis activity in MJ users. By examining existing studies we provide one of the first reviews aimed at synthesizing the literature on HPA axis functioning in MJ users.

Keywords: marijuana, hypothalamic-pituitary-adrenal axis, cortisol, adrenocorticotropic hormone, tetrahydrocannabinol

Introduction

Marijuana (MJ) is the most commonly used illicit substance worldwide, with ~147 million past year users (1). Within the United States, MJ use has significantly increased during the past decades. Between 2001 and 2002, 4.1% of adults reported past year MJ use compared to 9.5% between 2012 and 2013, 30% of whom met criteria for cannabis use disorder (2). These increases in MJ use coincide with a time of MJ decriminalization, legalization, and changing attitudes regarding risk of MJ use (3). While multiple risk factors contribute to heavy MJ use, cumulative stress is one pathway that may be linked to chronic MJ use as individuals report using MJ to reduce stress (4).

Several studies indicate that the hypothalamic-pituitary-adrenal (HPA) axis, the major neuroendocrine system that responds to stress (5), is dysregulated in substance users (6, 7). In response to acute stress as well as substances of abuse, the HPA axis releases corticotropin-releasing hormone (CRH) from the hypothalamus, which promotes release of adrenocorticotropic hormone (ACTH) from the anterior pituitary gland, ultimately resulting in cortisol secretion from the adrenal cortex. However, chronic stress and heavy substance use can lead to allostatic load, HPA axis dysfunction, and adverse effects on stress responsivity (8, 9). Despite the worldwide prevalence of MJ use, little is known about HPA axis response in heavy MJ users. Understanding how MJ use affects HPA axis functioning in humans is critical to informing studies on the role of the neuroendocrine stress system in chronic MJ users and in individuals at risk for heavy MJ use.

The purpose of this review is to provide a descriptive overview of prior research on the effects of acute MJ administration on HPA axis activity, the impact of stress on HPA axis functioning in MJ users, and the role of chronic MJ use on basal cortisol levels and the cortisol awakening response (CAR). Additionally, we compare methodological differences among studies that may have contributed to discrepant findings, and comment on future directions for advancing research in this field. Articles for this mini review were included based on combinations of keywords searched on Pubmed, including “marijuana”, “HPA axis”, “cortisol”, “tetrahydrocannabinol (THC)”, and “endocannabinoid.” Titles and abstracts were reviewed for relevance to the topic on human MJ users. Additional articles were found through citations included within the manuscripts found using the keyword search.

Effects of THC administration and acute marijuana use on HPA axis functioning in marijuana users

Several studies have investigated the effects of acute MJ administration on HPA axis response by examining ACTH and/or cortisol levels in a laboratory setting (Table ​ (Table1). 1 ). Cone et al. (11) found that MJ administration raised serum cortisol levels in MJ users compared to baseline. Similar results were reported by Kleinloog et al. (15), who reported that THC inhalation increased cortisol compared to baseline in infrequent MJ users. These findings were replicated in a resting state functional magnetic resonance imaging (fMRI) study, such that THC administration elevated cortisol levels compared to placebo in MJ users, but as there were no changes in hypothalamic connectivity observed, cortisol levels were not examined in relation to functional connectivity (16). In another study, De souse Fernades perna et al. (17) examined the effects of vaporized THC administration on cortisol response before and after an implicit association task displaying aggressive behavior, in which participants self-reported how aggressive they felt after viewing each image. MJ administration significantly elevated cortisol levels compared to placebo prior to aggression exposure. Cortisol levels were also higher after inhalation of MJ vs. unintoxicated baseline levels in an fMRI study examining the aphrodisiacal effects of MJ in MJ users. However, there were no differences in cortisol levels between MJ users with or without prior aphrodisiacal experiences, so its effect on brain activity was not examined further (18). Considering the lack of standardization of MJ administration, caution should be used when drawing conclusions based on these results. Overall, increases in cortisol after MJ administration may have both advantageous and disadvantageous effects. For example, as HPA axis activity mobilizes the body to face challenges, increased cortisol levels could be related to enhanced attention after acute MJ administration in heavy users (35), but could also be associated with impairments in other cognitive domains, such as working memory and inhibition (35), and increased anxiety (36). Thus, the increased cortisol response may be beneficial in certain contexts, but detrimental in others.

Table 1

Studies of HPA axis functioning in marijuana users.

Study Participants Sample size Age (mean ±SD) Study design ACTH or cortisol analysis Main findings
ACUTE MJ ADMINISTRATION STUDIES
Benowitz et al. (10) Regular MJ users (only male users) n = 6 21–30 210 mg oral THC for 14 days,.15 U/kg IV insulin Plasma cortisol No acute effect of THC on cortisol; following THC treatment, insulin administration ↓ cortisol compared to pre-THC treatment levels
Cone et al. (11) Frequent MJ users (only male users) n = 4 33.75 a Inhaled 2 MJ cigarettes (2.8% THC), 1 MJ and one placebo cigarette, or 2 placebo cigarettes (one condition per day) Plasma cortisol ↑ cortisol compared to baseline
Dax et al. (12) Abstinent heavy and occasional MJ users (only male users) n = 7 oral THC
n = 6 inhaled THC
n = 5 placebo
Age statistics not reported 10 mg oral THC (Marinol) or 18 mg/1.2 g inhaled MJ cigarette for 3 days (once on day 4) Plasma ACTH and cortisol No effect on cortisol for either method of administration
D’ Souza et al. (13) Abstinent MJ-dependent users and HC n = 30 MJ users
n = 22 HC
MJ = 24.8 ± 5.5
HC = 29 ± 11.6
0, 2.5, or 5 mg IV THC (one condition per day, test days separated by ≥1 week) Plasma cortisol Dose-dependent ↑ cortisol compared to placebo, blunted in frequent MJ users
Ranganathan et al. (14) Abstinent MJ-dependent users and HC n = 40 MJ users
n = 36 HC
MJ = 28.28 ± 10.2
HC = 24.58 ± 4.9
Study 1: Placebo, 0.0357 mg/kg, 0.0714 mg/kg IV THC Study 2: placebo, 0.0286 mg/kg IV THC Serum cortisol Dose-dependent ↑ cortisol, blunted in frequent MJ users
Kleinloog et al. (15) Mild MJ users (only male users) n = 49 mild MJ users 18–45 2-, 4-, and 6 mg inhaled THC at 90 min intervals Serum cortisol ↑ cortisol compared to baseline
Klumpers et al. (16) Occasional MJ users n = 12 22.17 ± 2.95 Day 1: 3 doses placebo Day 2: 2, 6, and 6 mg inhaled THC Serum cortisol ↑ cortisol compared to placebo
De Sousa Fernandes Perna et al. (17) Regular MJ users; heavy alcohol users; HC n = 21 regular MJ users
n = 20 alcohol users
n = 20 HC
MJ = 21.9 ± 2.2
Alcohol = 22.7 ± 2.4
HC = 22.9 ± 2.3
300 μg/kg vaporized MJ (12% THC), aggression implicit association task (IAT) Serum cortisol ↑ cortisol compared to placebo in MJ users administered THC prior to IAT
Androvicova et al. (18) Casual MJ users n = 12 aphrodisiac
n = 9 non-aphrodisiac
Aphrodisiac = 29.08 ± 5.37
Non-aphrodisiac = 23.78 ± 3.03
Inhaled socially relevant doses of personal MJ 30 min prior to study visit Serum cortisol ↑ cortisol compared to baseline
Childs et al. (19) occasional MJ users, ≤ 1 use per week n = 14: 0 mg THC
n = 15: 7.5 mg THC
n = 13: 12.5mg THC
0 mg THC = 23.8 ± 1.4
7.5 mg THC = 23.2 ± 0.9
12.5 mg THC = 23.9 ± 1.3
0, 7.5, or 12.5 mg oral THC (Marinol), Trier Social Stress Task Salivary cortisol No effect of THC on pre- or post- TSST cortisol levels
STRESS ADMINISTRATION STUDIES
Van Leeuwen et al. (20) Lifetime abstainers; lifetime tobacco users; and lifetime MJ users; users were also classified as repeated or lifetime users only n = 219 lifetime abstainers
n = 168 lifetime tobacco users
n = 204 lifetime MJ users
16.27 ± 0.73 Groningen Social Stress Test Salivary cortisol ↓ cortisol in lifetime MJ users vs. lifetime abstainers or lifetime tobacco users; similar finding in repeated MJ users vs. lifetime MJ or tobacco only users
Mcrae-Clark et al. (21) MJ-dependent n = 87 MJ stress group = 25.5 ± 9.2 MJ no stress group = 26.2 ± 8.0 Trier Social Stress Task; MJ cues Plasma ACTH and cortisol ↑ ACTH and cortisol in stress group; ↑ cortisol in response to neutral vs. MJ cues
Somaini et al. (22) Active MJ-dependent; abstinent MJ-dependent; HC n = 14 MJ-dependent
n = 14 abstinent MJ-dependent
n = 14 HC
MJ = 24.1 ± 2.7
HC = 25.4 ± 3.6
Unpleasant and neutral pictures from IAPS Plasma ACTH and cortisol Active MJ-dependent ↑ basal ACTH and cortisol vs. other groups, but smallest ↑ in ACTH and cortisol after viewing unpleasant images
Fox et al. (23) Treatment-seeking MJ, alcohol, cocaine dependent; alcohol/cocaine dependent; social drinking HC n = 30 MJ, alcohol, cocaine- dependent
n = 29 alcohol, cocaine- dependent
n = 26 social drinking HC
MJ/alcohol/cocaine = 33.7 ± 6.9
alcohol/cocaine = 37.1 ± 6.4
HC = 28.1 ± 1.4
Guided imagery (stress, alcohol/cocaine cue, relaxing) Plasma ACTH and cortisol MJ-dependent polysubstance users ↑ ACTH and cortisol to stress vs. relaxing imagery; effect not seen in other groups
Tull et al. (24) MJ-dependent PTSD; MJ-dependent no PTSD, PTSD only; no PTSD/no MJ-dependence n = 18 PTSD/MJ
n = 32 MJ no PTSD
n = 27 PTSD only
n = 91 no PTSD/no MJ
34.32 ± 10.1 Trauma cues Salivary cortisol No effect of trauma cues on cortisol
Cuttler et al. (25) Daily or near-daily MJ users; HC n = 40 daily MJ users
n = 42 HC
MJ users in stress condition = 26.05 ± 1.44
MJ users in no stress condition = 25.14 ± 1.86
HC in stress condition = 26.95 ± 2.23
HC in no stress condition = 25.24 ± 1.19 b
Maastricht Acute Stress Test Salivary cortisol ↓ cortisol in daily MJ users vs. HC
Nusbaum et al. (26) Daily or near-daily MJ users; HC n = 39 daily MJ users
n = 40 HC
MJ users in stress condition = 25.85 ± 6.19
MJ users in no stress condition = 25.35 ± 8.71
HC in stress condition = 27.25 ± 10.4
HC in no stress condition = 25.25 ± 5.57
Maastricht Acute Stress Test Salivary cortisol ↓ cortisol in daily MJ users vs. HC
Chao et al. (27) Non-treatment seeking daily MJ users with and without trauma exposure n = 125 Age range 18–50 (more detailed demographics of the six subgroups in table of manuscript) Trier Social Stress Task Salivary cortisol ↑ cortisol before, during, after TSST in daily MJ users with trauma exposure vs. daily MJ users without trauma exposure
BASAL CORTISOL STUDIES e
Block et al. (28) Frequent, moderate, infrequent, or non-Users of MJ n = 27 frequent MJ users
n = 18 moderate MJ users
n = 30 infrequent MJ users
n = 74 non-users
23.5 ± 0.4 b , more detailed demographics divided by user group and sex in table of manuscript Morning or afternoon blood draw Serum cortisol No difference between groups
King et al. (29) Daily or near daily MJ users; HC n = 30 MJ users
n = 30 HC
M MJ users = 21
F MJ users = 22.5
M HC = 23
F HC = 24.5 d
Morning saliva collection Salivary cortisol ↑ cortisol in MJ group compared to HC
Cloak et al. (30) Heavy MJ users; light MJ users; HC n = 43 heavy MJ users
n = 37 light MJ users
n = 42 HC
Heavy MJ users = 19.4 ± 0.3
Light MJ users = 19.1 ± 0.4
HC = 18.3 ± 0.4 c
Late morning or afternoon saliva collection f Salivary cortisol No difference between groups
Carol et al. (31) UHR youth with current MJ use; UHR youth without current MJ use; HC n = 17 UHR with MJ use
n = 26 UHR without MJ use
n = 29 HC
UHR with MJ use = 19.59 ± 0.87
UHR without MJ use = 18.46 ± 1.92
HC = 17.34 ± 2.82
Three saliva samples every 60 min between 8:45 a.m.−2 p.m. Salivary cortisol ↑ cortisol in MJ group compared to HC
Lisano et al. (32) Physically active regular MJ users; physical active HC (males only) n = 12 regular MJ users
n = 12 HC
MJ = 23.33 ± 4.14
HC = 24.08 ± 5.5
Blood samples collected between 7 and 9 a.m. Serum cortisol No difference between groups
CORTISOL AWAKENING RESPONSE STUDIES
Huizink et al. (33) Early (9–12 years old), late (13–14 years old), and non-users of MJ n = 59 late MJ users
n = 44 early MJ users
n = 1338 non-users
Cortisol collected between ages 10 and 12, age breakdown for groups not reported Saliva collected at awakening, 30 min later, and 8 pm Salivary cortisol ↓ cortisol 30 min post-awakening in early MJ users vs. late MJ users; MJ users ↑ evening cortisol vs. non-users
Montelone et al. (34) SCZ with MJ use prior to psychotic symptoms; SCZ with no MJ use prior to psychotic symptoms; HC n = 16 SCZ with MJ use
n = 12 SCZ without MJ use
n = 15 HC
SCZ with MJ use = 39.1 ± 7.2
SCZ without MJ use = 43.6 ± 7.3
HC = 37.6 ± 6.9 c
Saliva collected at awakening, and 15, 30, and 60 min later Salivary cortisol ↑ baseline cortisol in SCZ with MJ use vs. HC; ↓ CAR in SCZ with MJ use vs. HC

ACTH, adrenocorticotropic hormone; CAR, cortisol awakening response; HC, healthy controls; IV, intravenous; MJ, marijuana; PTSD, post-traumatic stress disorder; SCZ, schizophrenia; THC, tetrahydrocannabinol; TSST, Trier Social Stress Task; UHR, ultra-high risk; ↑, increase/increased/greater; ↓, decrease/decreased/less.

e As there was no specific drug administration or acute stress manipulation, study design refers to time of day for blood or saliva samples to measure basal cortisol levels.

f A subset of participants completed the TSST and computerized neuropsychological battery and saliva was also collected before and after these tests, but no effects were found.

In other studies, cortisol levels were compared between abstinent MJ-dependent individuals and non-users following intravenous THC administration. MJ-dependent individuals exhibited a blunted cortisol increase after THC administration compared to non-users (13, 14). Preclinical research has linked this blunted cortisol response to MJ tolerance (37), while other research suggests differences in MJ response may be influenced by genetics (38).

Some studies have found no significant effect of acute MJ administration on cortisol levels. In a study conducted by Benowitz et al. (10), the effects of insulin-induced hypoglycemia in MJ users were examined. Participants were given insulin prior to and after oral THC was administered. No difference in cortisol was observed between baseline and post-THC treatment but insulin administration decreased cortisol compared to pre-THC treatment levels. In a study by Dax et al. (12) abstinent MJ users were administered oral or inhaled THC. No differences were observed in ACTH or cortisol between baseline and post-treatment levels. Small sample sizes may have resulted in the lack of significant findings in these studies. Childs et al. (19) also found no relationship between acute administration of oral THC and cortisol response. The authors suggest their lack of findings could have been the result of collection of salivary rather than serum cortisol, the latter possibly being a more sensitive measure of cortisol. Moreover, in studies that found no effect of MJ on cortisol levels, greater time elapsed between MJ administration and cortisol assessment. It is possible the acute effects of MJ on cortisol could have diminished before cortisol assessment. Since studies that found an association between acute MJ administration and HPA axis response collected cortisol samples closer to the time of acute MJ use, it may be necessary for future studies to measure cortisol within 2 h following MJ administration. In sum, the majority of research examining acute MJ administration on cortisol reactivity has indicated MJ significantly increases cortisol, and some studies report that abstinent MJ-dependent users show a blunted increase in cortisol relative to non-users.

Effects of stress on HPA axis functioning in marijuana users

A number of studies in MJ users have examined the effects of acute stressors on HPA axis activity (Table ​ (Table1). 1 ). A study by Somaini et al. (22) presented neutral and unpleasant images to MJ-dependent individuals, abstinent MJ-dependent individuals, and healthy controls. Interestingly, active MJ users had generally high basal stress hormone levels but reduced responsivity of the HPA axis, potentially due to dysregulation of the stress system by MJ use. These findings are in contrast to another study in which MJ-dependent polysubstance users had significantly higher levels of plasma cortisol and ACTH following exposure to stress imagery relative to relaxing imagery, a finding not present in non-MJ-dependent polysubstance users or social drinkers (23). Since participants in this study were abstinent treatment-seeking polysubstance users, the elevated cortisol and ACTH levels could reflect a “rebound” upregulation of the HPA axis following abstinence (23).

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Two recent studies examined salivary cortisol in chronic adult MJ users using the Maastricht Acute Stress Test, which includes both physiological stress (placing hand in ice water) and psychosocial stress (solving math problems). The acute stress manipulation resulted in blunted cortisol response in the daily MJ users compared to healthy controls (25, 26). For individuals who may be characterized by an overactive HPA axis, a reduction in cortisol activity may be beneficial. Alternatively, cortisol release usually serves to motivate adaptive responses during stressful situations and a blunted response could impair one’s ability to act appropriately (8, 25). In particular, a blunted cortisol response to psychosocial stress has been associated with anxiety and depression in women (39), suggesting female MJ users may be at increased risk for anxiety and depression symptoms. Similar findings were reported in a study with a large sample (N = 591) of adolescent MJ users who had lifetime or repeated MJ use (20). The authors reported lower salivary cortisol levels during the Groningen Social Stress Task (involving both a speech and math problems) in adolescents who had ever used MJ relative to non-users or participants who reported lifetime tobacco use. This finding was also seen when the authors compared adolescents who used MJ at least five times in the past year with lifetime users of MJ or tobacco. These results were interpreted as a reduction in HPA axis response in adolescents who are at risk for using MJ repeatedly, possibly to stimulate their HPA axis response. Finally, another study that utilized the Trier Social Stress Task found that stress increased plasma ACTH and cortisol levels in MJ-dependent participants (21). However, in response to MJ cues, cortisol levels were significantly lower in MJ-dependent participants than in response to neutral cues. As the purpose of this study was to examine the effects of stress and drug cues on physiological reactivity in MJ-dependent individuals, no control group was included.

Other types of stress, such as previous trauma exposure, which may influence cortisol response in MJ users, was examined in a study of non-treatment seeking daily MJ users (27). Daily MJ users who experienced trauma had higher overall cortisol levels before, during, and after the Trier Social Stress Task than those who had never experienced trauma. However, as there was no control group of non-using participants in this study, it is uncertain whether the effects of trauma on cortisol reactivity would be similar to or different from the daily MJ users. Contrary to the findings of this study, Tull et al. (24) found no effects on cortisol reactivity in participants with or without post-traumatic stress disorder who were either MJ-dependent or non-dependent, even though MJ-dependent participants reported less subjective emotional reactivity in response to trauma cues.

Taken together, the findings to date suggest that stress exposure in adult heavy MJ users (22, 25, 26), or adolescents at risk for heavy MJ use (20) is mostly related to blunted reactivity of the HPA axis. This could suggest both dysregulation as result of MJ use or increased vulnerability toward frequent MJ use as individuals may engage in MJ use to increase responsivity of an underactive HPA axis. As there is currently limited research in this area, future studies should carefully consider the following variables, which could impact study findings: method of obtaining cortisol sample [plasma: (21–23) vs. saliva: (20, 24, 25, 27)], time of day of cortisol measurement, duration of time between stress administration and cortisol measurement, MJ use criteria (frequency of use, MJ-dependent or non-dependent sample, treatment seekers vs. non-treatment seekers), and co-occurring mental health conditions, such as previous trauma exposure (24, 27) or psychopathology (23).

Marijuana use and basal HPA axis activity

Studies have measured cortisol levels in frequent MJ users and non-users to determine whether the groups differ in basal cortisol levels, and findings suggest MJ has either no effect or increases basal cortisol (Table ​ (Table1). 1 ). Block et al. (28) found that there was no difference in serum cortisol levels between frequent, moderate, and infrequent MJ users and controls. However, only one blood sample was taken and time of day for blood draws varied among participants. There was also no difference in serum cortisol response in physically active MJ-using adults compared to non-using controls, suggesting that heavy MJ use may not affect stress hormone levels in individuals with high levels of physical activity (32). Since previous studies report that MJ may be used to reduce stress and anxiety symptoms (4), Cloak et al. (30) examined the relationship between MJ use, anxiety symptoms, and cortisol levels in adolescent and young adult heavy, light, and non-MJ users. There was no effect on mid-day salivary cortisol despite greater MJ use being associated with more anxiety symptoms, indicating a disconnect between psychological, and physiological stress reactivity.

Contrary to the findings above, an fMRI study examining psychomotor function found that chronic MJ users had higher levels of salivary cortisol compared with controls and greater superior frontal gyrus (SFG) but reduced visuomotor activity relative to controls (29). The authors propose that this increased cortisol in MJ users may impair visuomotor function during psychomotor tasks, resulting in greater reliance on brain regions involved in attention and motor planning, such as the SFG. A recent study of adolescents at ultra-high risk for schizophrenia reported that youth who used MJ in the past month had higher levels of salivary cortisol than healthy controls, suggesting a potential link between risk for psychosis and HPA axis functioning (31). Previous research indicates high basal cortisol levels are associated with hypertension and obesity (40), as well as hippocampal atrophy and memory impairment in aging populations (41). The potential effect of frequent MJ use on basal cortisol levels requires further investigation to clarify inconsistencies in the literature. Variations in participant characteristics, MJ use parameters, and method of cortisol assessment may have contributed to the inconsistent findings.

Cortisol awakening response in marijuana users

Cortisol levels exhibit diurnal variation, such that levels rise during the morning hours, peak 30 min after awakening, and are lowest in the evening. This increase of cortisol in the morning, known as the CAR is believed to be a reliable marker for individual differences in HPA axis activity (42). Studies have reported that the CAR is influenced by substance use, such as heavy alcohol use (43, 44). Surprisingly, little is known about the CAR in MJ users (Table ​ (Table1). 1 ). To our knowledge, only one study to date has examined diurnal cortisol response in MJ users, and found blunted levels of cortisol 30 min after awakening in a large sample of children (10–12 years old) who began using MJ during early adolescence (9–12 years old) relative to those who initiated use in later adolescence (13–14 years old) (33). These findings may indicate that blunted cortisol response could be a risk factor for initiating MJ use. The study also found that participants who initiated MJ use regardless of age at first use, had higher levels of evening cortisol relative to non-users. The authors believed this finding may be explained more by environmental influences on cortisol levels in the evening, such as ongoing stressful events rather than genetic vulnerability toward MJ use. Similar findings were reported in another study, albeit in a sample of participants diagnosed with schizophrenia who were also MJ users (34). These participants had higher baseline levels of cortisol, but a flattened CAR relative to healthy controls. These findings may indicate that MJ use in schizophrenics contributes to dysregulation of the HPA axis, although it is possible that blunted CAR in MJ-using schizophrenics predated and increased their vulnerability toward substance use. Given the lack of research investigating the CAR in MJ users, significant work is needed to characterize how MJ use affects the CAR and whether dysregulation of HPA axis functioning is a risk factor for and/or further drives MJ use.

Conclusions and future directions

The purpose of the current mini review is to highlight and integrate the existing, albeit limited literature on the effects of MJ use and stress on HPA axis functioning in adult MJ users and youth at risk for heavy MJ use. Understanding these findings comes at an important time when MJ decriminalization and legalization has made MJ increasingly available, while the perceived risk of MJ use has declined (3). Preclinical research has indicated that cannabinoids affect functioning of the HPA axis [for review, see Steiner and Wotjak (45)], and it appears that the current findings suggest that overall, acute MJ administration elevates cortisol levels, but to a smaller degree in MJ-dependent users. Further, acute stress exposure in heavy MJ users also appears to largely blunt cortisol reactivity. The findings on basal cortisol levels are mixed, likely due to diurnal fluctuations of cortisol and because cortisol is sensitive to changes in daily stress. These findings suggest that MJ use may dysregulate normal functioning of the HPA axis, perhaps as individuals develop tolerance to MJ, which could further drive MJ use. An alternative explanation is that individuals at risk for MJ use seek out MJ to stimulate an underactive HPA axis. Further research, including longitudinal studies of MJ users to examine long-term effects on HPA axis functioning are needed. While there is a growing literature on the effects of MJ use on brain structure and functioning in humans (46, 47), few studies have measured HPA axis activity in neuroimaging studies of MJ users, an important avenue for future research. This could provide information on how biological markers related to stress reactivity are associated with neurocognition in MJ users. Additionally, to our knowledge only two studies have examined the CAR in MJ-using participants, thus necessitating significant work in understanding how MJ use affects diurnal HPA axis rhythms. Work is currently underway in our laboratory to examine the effects of adult heavy MJ use on the CAR.

Author contributions

All authors contributed to the article search for this review. AC and JD-B wrote the Introduction, SL wrote the section on acute THC and MJ effects on HPA axis functioning, and AC wrote the sections on stress, basal cortisol, cortisol awakening response, and conclusions and future directions. AC and SL created the table. AC edited and finalized the manuscript.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work was supported by the Oregon State University Center for Humanities and College of Liberal Arts Research Grant to AC.

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