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. 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 (19). NAPE-PLD is a constitutively active enzyme involved in the biosynthesis of N-acylethanolamines, signaling lipids molecules like anandamide (20). 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 (21). 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 (22). In healthy volunteers, CBD enhances caudate and hippocampal activation and fronto-striatal connectivity during salience processing (23, 24) and under resting state conditions (25), enhances auditory and visual processing (26) and effects working and episodic memory associated with an increase in blood flow to the hippocampus (27). CBD alters functional coupling in cerebellum, frontal, and occipital cortices in patients with treatment resistant epilepsy (28) and attenuates hippocampal-striatal functional connectivity in psychosis patients (29). 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 (10, 11, 30, 31) and humans (32). The 10 mg/kg I.P. dose stood out as being the most effective, corroborating the many studies in rodents employing this dose (10-12, 30). 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 (33-45). 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 (46), its effects could be orchestrated easily by both peripheral and central targets.
CBD Targets
The primary targets for systemic CBD are unknown. Possible candidates include the cannabinoid CB1 receptors (47, 48), serotonin 5HT1a receptor, and the transient receptor potential vanilloid type 1 (TRPV1) (7, 49). 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 (18). 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 (50). 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 (51, 52) 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 (53-55). 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 (56, 57). 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 (58). However, CBD mediates the emotional and cardiovascular response to stress. CBD blunts the increased heart rate and blood pressure associated with restraint stress (12, 59, 60) and increase in blood pressure, heart rate and immobility behavior observed in contextual fear conditioning (61). CBD reduces immobility and escape behavior in mice exposed to a wild snake (62), altering the innate fear and aversion to predation. Rats exposed to cats present with long-lasting anxiogenic behavior that can be reduced with CBD (63). 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 (14), 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 (64). This last chance to escape predation is commonly referred to as death feigning (65). The immobility arises from descending neurons in the medullary, pontine reticular formation that suppress spinal motor neuron activity (66). The neural circuitry of tonic immobility includes much of the ARAS described here (67-69), in addition to the PAG (70, 71), basolateral and central amygdala (72), and medial dorsal thalamus (73). 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 (69, 74, 75). 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 (76). Indeed, the evidence for CBD and endocannabinoid signaling playing a significant role in emotional regulation in neuropsychiatric disorders is growing (77).
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.