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Endocannabinoid 2-arachidonoylglycerol (2-AG) has been implicated in habituation to stress, and its augmentation reduces stress-induced anxiety-like behavior. Chronic restraint stress (CRS) changes the 2-AG levels in some gross brain areas, such as the forebrain. However, the detailed spatial distribution of 2-AG and its changes by CRS in stress processing-related anatomical structures such as the anterior cingulate cortex (ACC), caudate putamen (CP), nucleus accumbens (NAc), and piriform cortex (PIR) are still unclear. In this study, mice were restrained for 30 min in a 50 mL-centrifuge tube for eight consecutive days, followed by imaging of the coronal brain sections of control and stressed mice using desorption electrospray ionization mass spectrometry imaging (DESI-MSI). The results showed that from the forebrain to the cerebellum, 2-AG levels were highest in the hypothalamus and lowest in the hippocampal region. 2-AG levels were significantly (p < 0.05) upregulated and 2-AG precursors levels were significantly (p < 0.05) downregulated in the ACC, CP, NAc, and PIR of stressed mice compared with control mice. This study provided direct evidence of 2-AG expression and changes, suggesting that 2-AG levels are increased in the ACC CP, NAc, and PIR when individuals are under chronic stress.

The role of endogenous cannabinoids in neuropathic pain has been actively studied, among which 2-arachidonoyl glycerol (2-AG) has received the most attention. However, owing to its chemical properties, direct detection of 2-AG distribution in tissues is difficult. Moreover, although desorption electrospray ionization mass spectrometry imaging (DESI-MSI) has enabled the detection of 2-AG, its distribution in the brain and spinal cord of neuropathic pain models has not been reported. In this study, the expression and distribution of 2-AG in the brain and spinal cord of a spare nerve injury (SNI) mice model of neuropathic pain was examined using DESI-MSI. The brain and lumbar spinal cord were collected and analyzed on days 3, 7, and 21 after treatment. On days 3 and 7 after treatment, 2-AG expression in the SNI model was decreased in the hypothalamus, midbrain, and especially in the periaqueductal gray (PAG) region but increased in the lumbar spinal cord. On day 21, the SNI model showed decreased 2-AG expression in the hypothalamus, but the difference from the control was not significant. Furthermore, there were no differences in 2-AG expression between the lumbar spinal cord, midbrain, or PAG. These data suggest that 2-AG might be involved in pain control.

The plasma membrane (PM) serves multiple functions to support cell activities with its heterogeneous molecular distribution. Fatty acids (FAs) are hydrophobic components of the PM whose saturation and length determine the membrane’s physical properties. The FA distribution contributes to the PM’s lateral heterogeneity. However, the distribution of PM FAs is poorly understood. Here, we proposed the FA cluster hypothesis, which suggested that FAs on the PM exist as clusters. By the optogenetic tool translocating the endoplasmic reticulum (ER), we were able to manipulate the distribution of PM FAs. We used time-of-flight combined secondary ion mass spectrometry (TOF-SIMS) to image PM FAs and discovered that PM FAs were presented and distributed as clusters and are also manipulated as clusters. We also found the existence of multi-FA clusters formed by the colocalization of more than one FA. Our optogenetic tool also decreased the clustering degree of FA clusters and the formation probability of multi-FA clusters. This research opens up new avenues and perspectives to study PM heterogeneity from an FA perspective. This research also suggests a possible treatment for diseases caused by PM lipid aggregation and furnished a convenient tool for therapeutic development.