A cannabinoid link between mitochondria and memory.

Cellular activity in the brain depends on the high energetic support provided by mitochondria, the cell organelles which use energy sources to generate ATP. Acute cannabinoid intoxication induces amnesia in humans and animals, and the activation of type-1 cannabinoid receptors present at brain mitochondria membranes (mtCB1) can directly alter mitochondrial energetic activity. Although the pathological impact of chronic mitochondrial dysfunctions in the brain is well established, the involvement of acute modulation of mitochondrial activity in high brain functions, including learning and memory, is unknown. Here, we show that acute cannabinoid-induced memory impairment in mice requires activation of hippocampal mtCB1 receptors. Genetic exclusion of CB1 receptors from hippocampal mitochondria prevents cannabinoid-induced reduction of mitochondrial mobility, synaptic transmission and memory formation. mtCB1 receptors signal through intra-mitochondrial Gαi protein activation and consequent inhibition of soluble-adenylyl cyclase (sAC). The resulting inhibition of protein kinase A (PKA)-dependent phosphorylation of specific subunits of the mitochondrial electron transport system eventually leads to decreased cellular respiration. Hippocampal inhibition of sAC activity or manipulation of intra-mitochondrial PKA signalling or phosphorylation of the Complex I subunit NDUFS2 inhibit bioenergetic and amnesic effects of cannabinoids. Thus, the G protein-coupled mtCB1 receptors regulate memory processes via modulation of mitochondrial energy metabolism. By directly linking mitochondrial activity to memory formation, these data reveal that bioenergetic processes are primary acute regulators of cognitive functions.

Estimation of reference intervals of five endocannabinoids and endocannabinoid related compounds in human plasma by two dimensional-LC/MS/MS.

The elucidation of the role of endocannabinoids in physiological and pathological conditions and the transferability of the importance of these mediators from basic evidence into clinical practice is still hampered by the indefiniteness of their circulating reference intervals. In this work, we developed and validated a two-dimensional LC/MS/MS method for the simultaneous measurement of plasma endocannabinoids and related compounds such as arachidonoyl-ethanolamide, palmitoyl-ethanolamide, and oleoyl-ethanolamide, belonging to the N-acyl-ethanolamide (NAE) family, and 2-arachidonoyl-glycerol and its inactive isomer 1-arachidonoyl-glycerol from the monoacyl-glycerol (MAG) family. We found that several pitfalls in the endocannabinoid measurement may occur, from blood withdrawal to plasma processing. Plasma extraction with toluene followed by on-line purification was chosen, allowing high-throughput and reliability. We estimated gender-specific reference intervals on 121 healthy normal weight subjects fulfilling rigorous anthropometric and hematic criteria. We observed no gender differences for NAEs, whereas significantly higher MAG levels were found in males compared with females. MAGs also significantly correlated with triglycerides. NAEs increased with age in females, and arachidonoyl-ethanolamide correlated with adiposity and metabolic parameters in females. This work paves the way to the establishment of definitive reference intervals for circulating endocannabinoids to help physicians move from the speculative research field into the clinical field.

Screening and Cellular Characterization of Genetically Encoded Voltage Indicators Based on Near-Infrared Fluorescent Proteins.

We developed genetically encoded voltage indicators using a transmembrane voltage-sensing domain and bright near-infrared fluorescent proteins derived from bacterial phytochromes. These new voltage indicators are excited by 640 nm light and emission is measured at 670 nm, allowing imaging in the near-infrared tissue transparency window. The spectral properties of our new indicators permit seamless voltage imaging with simultaneous blue-green light optogenetic actuator activation as well as simultaneous voltage-calcium imaging when paired with green calcium indicators. Iterative optimizations led to a fluorescent probe, here termed nirButterfly, which reliably reports neuronal activities including subthreshold membrane potential depolarization and hyperpolarization as well as spontaneous spiking or electrically- and optogenetically evoked action potentials. This enables largely improved all-optical causal interrogations of physiology.

Layer-specific potentiation of network GABAergic inhibition in the CA1 area of the hippocampus.

One of the most important functions of GABAergic inhibition in cortical regions is the tight control of spatiotemporal activity of principal neuronal ensembles. However, electrophysiological recordings do not provide sufficient spatial information to determine the spatiotemporal properties of inhibitory plasticity. Using Voltage Sensitive Dye Imaging (VSDI) in mouse hippocampal slices, we demonstrate that GABAA-mediated field inhibitory postsynaptic potentials undergo layer-specific potentiation upon activation of metabotropic glutamate receptors (mGlu). VSDI recordings allowed detection of pharmacologically isolated GABAA-dependent hyperpolarization signals. Bath-application of the selective group-I mGlu receptor agonist, (S)-3,5-Dihydroxyphenylglycine (DHPG), induces an enhancement of the GABAergic VSDI-recorded signal, which is more or less pronounced in different hippocampal layers. This potentiation is mediated by mGlu5 and downstream activation of IP3 receptors. Our results depict network GABAergic activity in the hippocampal CA1 region and its sub-layers, showing also a novel form of inhibitory synaptic plasticity tightly coupled to glutamatergic activity.