Izaskun Elezgarai

Molecular components and functions of the endocannabinoid system in mouse prefrontal cortex.

Background Cannabinoids have deleterious effects on prefrontal cortex (PFC)-mediated functions and multiple evidences link the endogenous cannabinoid (endocannabinoid) system, cannabis use and schizophrenia, a disease in which PFC functions are altered. Nonetheless, the molecular composition and the physiological functions of the endocannabinoid system in the PFC are unknown. Methodology/Principal Findings Here, using electron microscopy we found that key proteins involved in endocannabinoid signaling are expressed in layers V/VI of the mouse prelimbic area of the PFC: presynaptic cannabinoid CB1 receptors (CB1R) faced postsynaptic mGluR5 while diacylglycerol lipase α (DGL-α), the enzyme generating the endocannabinoid 2-arachidonoyl-glycerol (2-AG) was expressed in the same dendritic processes as mGluR5. Activation of presynaptic CB1R strongly inhibited evoked excitatory post-synaptic currents. Prolonged synaptic stimulation at 10Hz induced a profound long-term depression (LTD) of layers V/VI excitatory inputs. The endocannabinoid -LTD was presynaptically expressed and depended on the activation of postsynaptic mGluR5, phospholipase C and a rise in postsynaptic Ca2+ as predicted from the localization of the different components of the endocannabinoid system. Blocking the degradation of 2-AG (with URB 602) but not of anandamide (with URB 597) converted subthreshold tetanus to LTD-inducing ones. Moreover, inhibiting the synthesis of 2-AG with Tetrahydrolipstatin, blocked endocannabinoid-mediated LTD. All together, our data show that 2-AG mediates LTD at these synapses. Conclusions/Significance Our data show that the endocannabinoid -retrograde signaling plays a prominent role in long-term synaptic plasticity at the excitatory synapses of the PFC. Alterations of endocannabinoid -mediated synaptic plasticity may participate to the etiology of PFC-related pathologies.

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.

Immunolocalization of the mGluR1b Splice Variant of the Metabotropic Glutamate Receptor 1 at Parallel Fiber-Purkinje Cell Synapses in the Rat Cerebellar Cortex

Several metabotropic glutamate receptor (mGluR) subtypes have been identified in the cerebellar cortex that are targeted to different compartments in cerebellar cells. In this study, preembedding immunocytochemical methods for electron microscopy were used to investigate the subcellular distribution of the mGluR1b splice variant in the rat cerebellar cortex. Dendritic spines of Purkinje cells receiving parallel fiber synaptic terminals were immunoreactive for mGluR1b. With a preembedding immunogold method, ~25% of the mGluR1b immunolabeling was observed perisynaptically within 60 nm from the edge of the postsynaptic densities. Values of extrasynaptic gold particles beyond the first 60 nm were maintained at between 10 and 18% along the whole intracellular surface of the dentritic spine membranes of Purkinje cells. For comparison, the distribution of mGluR1a was studied. A predominant (~37%) perisynaptic localization of mGluR1a was seen in dendritic spines of Purkinje cells, dropping the extrasynaptic labeling to 15% in the 60‐120‐nm bin from the edge of the postsynaptic specialization. Our results reveal that mGluR1b and mGluR1a are localized to the same subcellular compartments in Purkinje cells but that the densities of the perisynaptic and extrasynaptic pools were different for both isoforms. The compartmentalization of mGluR1b and mGluR1a might serve distinct requirements in cerebellar neurotransmission.

Cannabinoid Receptors in the Bed Nucleus of the Stria Terminalis Control Cortical Excitation of Midbrain Dopamine Cells In Vivo

The endocannabinoid system is involved in multiple physiological functions including reward. Cannabinoids potently control the activity of midbrain dopamine cells, but the contribution of cortical projections in this phenomenon is unclear. We show that the bed nucleus of the stria terminalis (BNST) efficient relays cortical excitation to dopamine neurons of the ventral tegmental area (VTA). Anatomical and in vivo electrophysiological evidence demonstrate that excitatory projections arising exclusively from the infralimbic cortex converge on BNST neurons, which in turn project to and excite >80% VTA dopamine cells. At the ultrastructural level, cannabinoid type 1 receptors are detected within the BNST on axon terminals arising from the infralimbic cortex. We found that intra-BNST infusion of a cannabinoid agonist inhibits the firing of dopamine cells evoked by stimulation of the infralimbic cortex. Our data identify a new neuronal substrate for the actions of cannabinoids in the reward pathway.

Immunoreactivity for the group III metabotropic glutamate receptor subtype mGluR4a in the superficial laminae of the rat spinal dorsal horn

Studies indicate that metabotropic glutamate receptors (mGluRs) may play a role in spinal sensory transmission. We examined the cellular and subcellular distribution of the mGluR subtype 4a in spinal tissue by means of a specific antiserum and immunocytochemical techniques for light and electron microscopy. A dense plexus of mGluR4a‐immunoreactive elements was seen in the dorsal horn, with an apparent accumulation in lamina II. The immunostaining was composed of sparse immunoreactive fibres and punctate elements. No perikaryal staining was seen. Immunostaining for mGluR4a was detected in small to medium‐sized cells but not in large cells in dorsal root ganglia. At the electron microscopic level, superficial dorsal horn laminae demonstrated numerous immunoreactive vesicle‐containing profiles. Labelling was present in the cytoplasmic matrix, but accretion of immunoreaction product to presynaptic specialisations was commonly observed. Axolemmal labelling was confirmed by using a preembedding immunogold technique, which revealed distinctive deposits of gold immunoparticles along presynaptic thickenings with an average centre‐to‐centre distance of 41 nm (41.145 ± 13.59). Immunoreactive terminals often formed synaptic contacts with dendritic profiles immunonegative for mGluR4a. Immunonegative dendritic profiles were observed in apposition to both mGluR4a‐immunoreactive and immunonegative terminals. Diffuse immunoperoxidase reaction product was also detected in dendritic profiles, some of which were contacted by mGluR4a‐immunoreactive endings, but only occassionally were they observed to accumulate immunoreaction product along the postsynaptic density. Terminals immunoreactive for mGluR4a also formed axosomatic contacts. The present results reveal that mGluR4a subserves a complex spinal circuitry to which the primary afferent system seems to be a major contributor. J. Comp. Neurol. 430:448–457, 2001.

DEVELOPMENTAL EXPRESSION OF THE GROUP III METABOTROPIC GLUTAMATE RECEPTOR MGLUR4A IN THE MEDIAL NUCLEUS OF THE TRAPEZOID BODY OF THE RAT

A preembedding immunocytochemical method for light microscopy was used to study the postnatal development of expression of the group III metabotropic glutamate receptor mGluR4a in the medial nucleus of the trapezoid body (MNTB) of the rat. Immunoreactivity for mGluR4a was localized in axonal endings wrapping the principal globular neurons in MNTB, known as calyces of Held. The percentage of calyces of Held immunoreactive for mGluR4a increased progressively from postnatal day 3 (PND3), showing the highest density of labeled calyces by PND9. From this postnatal age on, a gradual reduction in the number of mGluR4a‐immunopositive calyces of Held was observed, reaching the lowest level of labeled profiles in adult tissue. The developmental expression of mGluR4a in calyces of Held correlates well with previous studies in young animals showing a modulation of synaptic neurotransmission by group III mGluRs in these giant excitatory synapses made on MNTB principal neurons. All these observations together suggest that the expression of mGluR4a mainly between PND7 and PND12 might be relevant to the maturation and modulation of synaptic transmission at the calyces of Held. J. Comp. Neurol. 411:431–440, 1999.

The metabotropic glutamate receptor subtype mGluR 2/3 is located at extrasynaptic loci in rat spinal dorsal horn synapses

The position of neurotransmitter receptors relative to active neurotransmitter release sites may be a major factor influencing neuronal responses. The location of the metabotropic glutamate receptor subtype mGluR2/3 was investigated in synaptic structures in the rat superficial spinal dorsal horn laminae by using a pre-embedding immunogold technique. Immunostaining for mGluR2/3 occurred in laminae I through III. Gold particles were encountered both in the cytosol and along the plasma membrane. Distinctive plasmalemmal immunodeposits were detected in vesicle-containing profiles, where they were located to membrane compartments distant from active release sites rather than in the close vicinity of synaptic specialisations. No distinct immunolabelling was observed in profiles meeting characteristics of primary afferent terminals. The extrasynaptic occurrence of mGluR2/3 suggests a presynaptic heteroreceptor role for these receptor subtypes in the spinal dorsal horn.

Clustering of the group III metabotropic glutamate receptor 4a at parallel fiber synaptic terminals in the rat cerebellar molecular layer.

We report in this study with a pre-embedding immunogold method, the clustering of the group III metabotropic glutamate receptor 4a (mGluR4a) along the presynaptic membrane of parallel fiber synaptic terminals in the cerebellar molecular layer. The mGluR4a clusters were homogeneously distributed and interspaced by about 60 nm. These results suggest a particular arrangement of mGluR4a which might help to a rapid and effective activation of this receptor by glutamate.

Localization and Function of the Cannabinoid CB1 Receptor in the Anterolateral Bed Nucleus of the Stria Terminalis

Background The bed nucleus of the stria terminalis (BNST) is involved in behaviors related to natural reward, drug addiction and stress. In spite of the emerging role of the endogenous cannabinoid (eCB) system in these behaviors, little is known about the anatomy and function of this system in the anterolateral BNST (alBNST). The aim of this study was to provide a detailed morphological characterization of the localization of the cannabinoid 1 (CB1) receptor a necessary step toward a better understanding of the physiological roles of the eCB system in this region of the brain. Methodology/Principal Findings We have combined anatomical approaches at the confocal and electron microscopy level to ex-vivo electrophysiological techniques. Here, we report that CB1 is localized on presynaptic membranes of about 55% of immunopositive synaptic terminals for the vesicular glutamate transporter 1 (vGluT1), which contain abundant spherical, clear synaptic vesicles and make asymmetrical synapses with alBNST neurons. About 64% of vGluT1 immunonegative synaptic terminals show CB1 immunolabeling. Furthermore, 30% and 35% of presynaptic boutons localize CB1 in alBNST of conditional mutant mice lacking CB1 mainly from GABAergic neurons (GABA-CB1-KO mice) and mainly from cortical glutamatergic neurons (Glu-CB1-KO mice), respectively. Extracellular field recordings and whole cell patch clamp in the alBNST rat brain slice preparation revealed that activation of CB1 strongly inhibits excitatory and inhibitory synaptic transmission. Conclusions/Significance This study supports the anterolateral BNST as a potential neuronal substrate of the effects of cannabinoids on stress-related behaviors.

Targeting the endocannabinoid system : future therapeutic strategies

The endocannabinoid system (ECS) is involved in many physiological regulation pathways in the human body, which makes this system the target of many drugs and therapies. In this review, we highlight the latest studies regarding the role of the ECS and the drugs that target it, with a particular focus on the basis for the discovery of new cannabinoid-based drugs. In addition, we propose some key steps, such as the creation of a cannabinoid-receptor interaction matrix (CRIM) and the use of metabolomics, toward the development of improved and more specific drugs for each relevant disease.