GLAST versus GFAP as astroglial marker for the subcellular study of cannabinoid CB1 receptors in astrocytes.

The cannabinoid CB1 receptor-mediated functions in astrocytes are highly dependent on the CB1 receptor distribution in these glial cells relative to neuronal sites, particularly at the nearby synapses under normal or pathological conditions. However, the portrait of the CB1 receptor distribution in astroglial compartments remains uncompleted because of the scarce CB1 receptor expression in these cells and the limited identification of astrocytes. The glial fibrillary acidic protein (GFAP) is commonly used as astroglial marker. However, because GFAP is a cytoskeleton protein mostly restricted to the astroglial cell bodies and their main branches, it seems not ideal for the localization of CB1 receptor distribution in astrocytes. Therefore, alternative markers to decipher the actual astroglial CB1 receptors are required. In this work, we have compared the glutamate aspartate transporter (GLAST) versus GFAP for the CB1 receptor localization in astrocytes. We found by immunoelectron microscopy that GLAST reveals almost three-fold astroglial area and four-fold astroglial membranes compared to GFAP. In addition, this better visualization of astrocytes was associated with the detection of 12% of the total CB1 receptor labeling in GLAST-positive astrocytes.

Lack of the Transient Receptor Potential Vanilloid 1 Shifts Cannabinoid-Dependent Excitatory Synaptic Plasticity in the Dentate Gyrus of the Mouse Brain Hippocampus.

The transient receptor potential vanilloid 1 (TRPV1) participates in synaptic functions in the brain. In the dentate gyrus, post-synaptic TRPV1 in the granule cell (GC) dendritic spines mediates a type of long-term depression (LTD) of the excitatory medial perforant path (MPP) synapses independent of pre-synaptic cannabinoid CB1 receptors. As CB1 receptors also mediate LTD at these synapses, both CB1 and TRPV1 might be influencing the activity of each other acting from opposite synaptic sites. We tested this hypothesis in the MPP-GC synapses of mice lacking TRPV1 (TRPV1-/-). Unlike wild-type (WT) mice, low-frequency stimulation (10 min at 10 Hz) of TRPV1-/- MPP fibers elicited a form of long-term potentiation (LTP) that was dependent on (1) CB1 receptors, (2) the endocannabinoid 2-arachidonoylglycerol (2-AG), (3) rearrangement of actin filaments, and (4) nitric oxide signaling. These functional changes were associated with an increase in the maximum binding efficacy of guanosine-5'-O-(3-[35S]thiotriphosphate) ([35S]GTPγS) stimulated by the CB1 receptor agonist CP 55,940, and a significant decrease in receptor basal activation in the TRPV1-/- hippocampus. Finally, TRPV1-/- hippocampal synaptosomes showed an augmented level of the guanine nucleotide-binding (G) Gαi1, Gαi2, and Gαi3 protein alpha subunits. Altogether, the lack of TRPV1 modifies CB1 receptor signaling in the dentate gyrus and causes the shift from CB1 receptor-mediated LTD to LTP at the MPP-GC synapses.

Glucose metabolism links astroglial mitochondria to cannabinoid effects.

Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses1-5. By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors5-7. However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB1) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifically, activation of astroglial mtCB1 receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and affects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these effects abolishes the effect of cannabinoid treatment on the observed behaviour. These findings suggest that mtCB1 receptor signalling can directly regulate astroglial glucose metabolism to fine-tune neuronal activity and behaviour in mice.

Cannabidiol Administration Prevents Hypoxia-Ischemia-Induced Hypomyelination in Newborn Rats.

Neonatal hypoxia-ischemia (HI) is a risk factor for myelination disturbances, a key factor for cerebral palsy. Cannabidiol (CBD) protects neurons and glial cells after HI insult in newborn animals. We hereby aimed to study CBD's effects on long-lasting HI-induced myelination deficits in newborn rats. Thus, P7 Wistar rats received s.c. vehicle (HV) or cannabidiol (HC) after HI brain damage (left carotid artery electrocoagulation plus 10% O2 for 112 min). Controls were non-HI pups. At P37, neurobehavioral tests were performed and immunohistochemistry [quantifying mature oligodendrocyte (mOL) populations and myelin basic protein (MBP) density] and electron microscopy (determining axon number, size, and myelin thickness) studies were conducted in cortex (CX) and white matter (WM). Expression of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) were analyzed by western blot at P14. HI reduced mOL or MBP in CX but not in WM. In both CX and WM, axon density and myelin thickness were reduced. MBP impairment correlated with functional deficits. CBD administration resulted in normal function associated with normal mOL and MBP, as well as normal axon density and myelin thickness in all areas. CBD's effects were not associated with increased BDNF or GDNF expression. In conclusion, HI injury in newborn rats resulted in long-lasting myelination disturbance, associated with functional impairment. CBD treatment preserved function and myelination, likely as a part of a general neuroprotective effect.

CB1 Receptors in the Anterior Piriform Cortex Control Odor Preference Memory.

The retrieval of odor-related memories shapes animal behavior. The anterior piriform cortex (aPC) is the largest part of the olfactory cortex, and it plays important roles in olfactory processing and memory. However, it is still unclear whether specific cellular mechanisms in the aPC control olfactory memory, depending on the appetitive or aversive nature of the stimuli involved. Cannabinoid-type 1 (CB1) receptors are present in the aPC (aPC-CB1), but their potential impact on olfactory memory was never explored. Here, we used a combination of behavioral, genetic, anatomical, and electrophysiological approaches to characterize the functions of aPC-CB1 receptors in the regulation of appetitive and aversive olfactory memory. Pharmacological blockade or genetic deletion of aPC-CB1 receptors specifically impaired the retrieval of conditioned odor preference (COP). Interestingly, expression of conditioned odor aversion (COA) was unaffected by local CB1 receptor blockade, indicating that the role of aPC endocannabinoid signaling is selective for retrieval of appetitive memory. Anatomical investigations revealed that CB1 receptors are highly expressed on aPC GABAergic interneurons, and ex vivo electrophysiological recordings showed that their pharmacological activation reduces miniature inhibitory post-synaptic currents (mIPSCs) onto aPC semilunar (SL), but not pyramidal principal neurons. COP retrieval, but not COA, was associated with a specific CB1-receptor-dependent decrease of mIPSCs in SL cells. Altogether, these data indicate that aPC-CB1 receptor-dependent mechanisms physiologically control the retrieval of olfactory memory, depending on odor valence and engaging modulation of local inhibitory transmission.

Endocannabinoid long-term depression revealed at medial perforant path excitatory synapses in the dentate gyrus.

The endocannabinoid system modulates synaptic plasticity in the hippocampus, but a link between long-term synaptic plasticity and the type 1 cannabinoid (CB1) receptor at medial perforant path (MPP) synapses remains elusive. Here, immuno-electron microscopy in adult mice showed that ∼26% of the excitatory synaptic terminals in the middle 1/3 of the dentate molecular layer (DML) contained CB1 receptors, and field excitatory postsynaptic potentials evoked by MPP stimulation were inhibited by CB1 receptor activation. In addition, MPP stimulation at 10 Hz for 10 min triggered CB1 receptor-dependent excitatory long-term depression (eCB-eLTD) at MPP synapses of wild-type mice but not on CB1-knockout mice. This eCB-eLTD was group I mGluR-dependent, required intracellular calcium influx and 2-arachydonoyl-glycerol (2-AG) synthesis but did not depend on N-methyl-d-aspartate (NMDA) receptors. Overall, these results point to a functional role for CB1 receptors with eCB-eLTD at DML MPP synapses and further involve these receptors in memory processing within the adult brain.

High-resolution Immunoelectron Microscopy Techniques for Revealing Distinct Subcellular Type 1 Cannabinoid Receptor Domains in Brain.

Activation of type 1 cannabinoid (CB1) receptors by endogenous, exogenous (cannabis derivatives) or synthetic cannabinoids (i.e., CP 55.940, Win-2) has a wide variety of behavioral effects due to the presence of CB1 receptors in the brain. In situ hybridization and immunohistochemical techniques have been crucial for defining the CB1 receptor expression and localization at the cellular level. Nevertheless, more advanced methods are needed to reveal the precise topography of CB1 receptors in the brain, especially in unsuspected sites such as other cell types and organelles with low receptor expression (e.g., glutamatergic neurons, astrocytes, mitochondria). High-resolution immunoelectron microscopy provides a more precise detection method for the subcellular localization of CB1 receptors in the brain. Herein, we describe a single pre-embedding immunogold method for electron microscopy based on the use of specific CB1 receptor antibodies and silver-intensified 1.4 nm gold-labeled Fab' fragments, and a combined pre-embedding immunogold and immunoperoxidase method that employs biotinylated secondary antibodies and avidin-biotin-peroxidase complex for the simultaneous localization of CB1 receptors and protein markers of specific brain cells or synapses (e.g., GFAP, GLAST, IBA-1, PSD-95, gephyrin). In addition, a post-embedding immunogold method is also described and compared to the pre-embedding labeling procedure. These methods provide a relatively easy and useful approach for revealing the subcellular localization of low amounts of CB1 receptors in glutamatergic synapses, astrocytes, neuronal and astrocytic mitochondria in the brain.