Many have heard of cannabinoids – parts of marijuana that act in the brain and cause the cluster of effects identified as the ‘high’. Cannabinoids, like other drugs, work in the brain by engaging a receptor; essentially acting like the key to a lock. Humans feel the effects of marijuana because the brain is replete with receptors for cannabinoids. Brain cannabinoid receptors are numerous, not in order to respond to the psychedelic elements of cannabis plants, but to respond to endogenous cannabinoid compounds made by the brain called endocannabinoids.



Endocannabinoids act unconventionally in the brain by traveling backwards. In general, signaling between the brain’s neurons is in one direction; a chemical signal is sent from the axon terminal of one neuron, this neurotransmitter crosses the synapse between neurons and then engages a receptor on the postsynaptic neuron. Endocannabinoids are released from the postsynaptic neuron, travel retrogradely across the synapse and engage a receptor on the presynaptic site. This type of signaling is very powerful. It is a way in which the postsynaptic neuron can listen to signals sent by a presynaptic neuron and send signals back to modify the presynaptic neuron’s behavior. Essentially, this allows neurons to have a conversation. Instead of simply receiving a message and passing it along, with retrograde signaling the postsynaptic neuron can reply to the first, telling it that it’d like to receive less signal.



The CB1 cannabinoid receptor is one of the most copiously expressed presynaptic receptors in the brain. Despite its abundance, its distribution is still poorly understood, although it is generally known to be found on inhibitory interneurons in the hippocampus (a brain formation with well-known connection to memory processing). Axons from interneurons have two important kinds of connections to post-synaptic neurons: either strongly effective connections to the cell body (axo-somatic) or to the weaker connections to the receptive dendritic branches (axo-dendritic). These kinds of connections are effected differently by cannabinoids. Low doses of cannabinoids prevent the release of the inhibitory neurotransmitter GABA from axo-somatic connections but not from axo-dendritic connections. Adding complexity, CB1 receptors can be found on the neuronal surface or the intracellular compartment. Additionally, retrograde signaling to cannabinoid receptors is more likely to reduce the release of the inhibitory neurotransmitter GABA than the excitatory neurotransmitter glutamate. Changes in the distribution and function of CB1 are associated with epilepsy and Fragile X syndrome in mice. Clearly, the effect of endocannabinoids and cannabinoids depends on the type of cells and connections where the CB1 receptor is found, but until now this could not be seen with sufficient resolution using traditional microscopy techniques.



In order to understand the distribution of presynaptic CB1 receptors in a way that would give insight into its role in endocanibinoid signaling and in signaling after use of marijuana, Dudok and an colleagues used a new technique called stochastic optical reconstruction microscopy (STORM) to visualize CB1 receptor location in super-resolution in mouse brains. The findings are published online ahead of print in Nature Neuroscience. To put this in perspective, literally and figuratively, the authors visualized CB1 receptors in synapses on a nanometer scale, whereas traditional imaging could only provide resolution on a micrometer scale. Additionally, the authors combined the STORM imaging with a patch-clamp technique that allowed them to record responses from individual cells, fill them with a dye and observe the distribution of CB1 receptors and other target proteins on those cells with confocal microscopy. This combination is a unique way to capture both physiological and morphological data from the same cell with high temporal and spatial resolution.



With this technique, the investigators were able to view CB1 receptors on individual neurons within groups of different types of neurons and characterize the distribution of CB1 receptors on the cell surface and located in the intracellular compartment. They quantified the distance between CB1 receptors and synaptic machinery that releases vesicles filled with neurotransmitter to communicate with the post-synaptic site. The authors suggest that the distance of CB1 receptors from this machinery is likely to determine the strength at which retrograde endocannabinoid signaling is able to reduce presynaptic signaling.



They found that CB1 receptors are spread homogenously on the pre-synaptic terminal and that there are more CB1 receptors on larger axo-somatic terminals which had proportionately less of the important cellular machinery for neurotransmitter release. Because there are more CB1 receptors relative to this machinery, this may explain why axo-somatic synapses respond more sensitively to low concentrations of endocannabinoid signaling, whereas both synapse types respond similarly to endocannabinoid levels that saturate the CB1 receptors.



The authors then manipulated this system by treating mice with Δ9-tetrahydrocannabinol (THC, the main psychoactive component of marijuana). They chose a low-dose that is similar to what humans use for medicinal purposes, and a higher dose roughly equivalent to humans smoking marijuana with low THC content. The higher dose is associated with behavioral tolerance and a change in the pattern of CB1 receptor distribution. They evaluated some mice after 6 weeks of treatment and some after a combined treatment and withdrawal period.



The low dose of THC did not change CB1 receptors much, but the higher dose associated with tolerance drove the loss of pre-synaptic CB1 receptors and the internalization of remaining CB1 receptors. Fewer pre-synaptic CB1 receptors on the surface after chronic exposure to THC would explain the reduced ability of endocannabinoids to supress GABA release from inhibitory interneurons. CB1 receptors returned to normal after 6 weeks withdrawal.



This study combined two sophisticated techniques in order to see cannabinoid receptor distribution on a new scale with incredible resolution. This approach will open doorways to evaluation of other synaptic proteins on this level. Additionally, the authors used this technique to give clarity to the action of cannabinoids at levels associated with medical use and recreational use, and timing associated with current use and past use. These findings cross a barrier for cellular and molecular research and also will open doors for research on cannabinoid and endocannabinoid function.