We developed an advanced clearing method with superior fluorescence-preserving capability for 3D imaging of whole organs.

Endocannabinoids (eCBs) are endogenous lipid mediators involved in a variety of physiological, pharmacological, and pathological processes. While activation of the eCB system primarily induces inhibitory effects on both GABAergic and glutamatergic synaptic transmission and plasticity through acting on presynaptically-expressed CB1 receptors in the brain, accumulated information suggests that eCB signaling is also capable of facilitating or potentiating excitatory synaptic transmission in the hippocampus. Recent studies show that a long-lasting potentiation of excitatory synaptic transmission at Schaffer collateral (SC)-CA1 synapses is induced by spatiotemporally primed inputs, accompanying with a long-term depression of inhibitory synaptic transmission (I-LTD) in hippocampal CA1 pyramidal neurons. This input-timing-dependent long-lasting synaptic potentiation at SC-CA1 synapses is mediated by 2-arachidonoylglycerol (2-AG) signaling triggered by activation of postsynaptic NMDA receptors, group I metabotropic glutamate receptors (mGluRs), and a concurrent rise in intracellular Ca2+. Emerging evidence now also indicates that 2-AG is an important signaling mediator keeping brain homeostasis by exerting its anti-inflammatory and neuroprotective effects in response to harmful insults through CB1/2 receptor-dependent and/or independent mechanisms. Activation of the nuclear receptor protein peroxisome proliferator-activated receptor-γ (PPARγ) apparently is one of the important mechanisms in resolving neuroinflammation and protecting neurons produced by 2-AG signaling. Thus, the information summarized in this review suggests that the role of eCB signaling in maintaining integrity of brain function is greater than what we thought previously.

Recently, a variety of tissue optical clearing techniques have been developed to reduce light scattering for imaging deeper and three-dimensional reconstruction of tissue structures. Combined with optical imaging techniques and diverse labeling methods, these clearing methods have significantly promoted the development of neuroscience. Each of them has its own characteristics with certain advantages and disadvantages. Though there are some comparison results, the clearing methods covered are limited and the evaluation indices lack uniformity, which made it difficult to select a best-fit protocol from numerous methods for clearing in practical applications. Hence, it is necessary to systematically assess and compare these clearing methods. We evaluated the performance of seven typical clearing methods, including 3-D imaging of solvent-cleared organs (3DISCO), ultimate DISCO (uDISCO), see deep brain (SeeDB), ScaeS, , clear, unobstructed brain imaging cocktails and computational analysis, and passive CLARITY technique (PACT), on mouse brain samples. First, we compared the clearing effect and clearing time as well as size deformation on brain tissues. Further, we evaluated the fluorescence preservation and the increase of imaging depth induced by different methods. The results showed that 3DISCO, uDISCO, and PACT possessed excellent clearing capability on mouse brains, ScaeS and SeeDB rendered moderate transparency, whereas performed the worst. uDISCO and 3DISCO induced substantial size reduction on brain sections, and PACT expanded the mouse brain most seriously. Among those methods, ScaeS performed best on fluorescence retention, 3DISCO induced the biggest decline of the fluorescence. PACT achieved the highest increase of imaging depth, and SeeDB and possessed the shallowest imaging depth. This study is expected to provide important reference for users in choosing the most suitable brain optical clearing method.