Effects of Cannabidiol, Hypothermia, and Their Combination in Newborn Rats with Hypoxic-Ischemic Encephalopathy

Therapeutic hypothermia is well established as a standard treatment for infants with hypoxic-ischemic (HI) encephalopathy but it is only partially effective. The potential for combination treatments to augment hypothermic neuroprotection has major relevance. Our aim was to assess the effects of treating newborn rats following HI injury with cannabidiol (CBD) at 0.1 or 1 mg/kg, i.p., in normothermic (37.5°C) and hypothermic (32.0°C) conditions, from 7 d of age (neonatal phase) to 37 d of age (juvenile phase). Placebo or CBD was administered at 0.5, 24, and 48 h after HI injury. Two sensorimotor (rotarod and cylinder rearing) and two cognitive (novel object recognition and T-maze) tests were conducted 30 d after HI. The extent of brain damage was determined by magnetic resonance imaging, histologic evaluation, magnetic resonance spectroscopy, amplitude-integrated electroencephalography, and Western blotting. At 37 d, the HI insult produced impairments in all neurobehavioral scores (cognitive and sensorimotor tests), brain activity (electroencephalography), neuropathological score (temporoparietal cortexes and CA1 layer of hippocampus), lesion volume, magnetic resonance biomarkers of brain injury (metabolic dysfunction, excitotoxicity, neural damage, and mitochondrial impairment), oxidative stress, and inflammation (TNFα). We observed that CBD or hypothermia (to a lesser extent than CBD) alone improved cognitive and motor functions, as well as brain activity. When used together, CBD and hypothermia ameliorated brain excitotoxicity, oxidative stress, and inflammation, reduced brain infarct volume, lessened the extent of histologic damage, and demonstrated additivity in some parameters. Thus, coadministration of CBD and hypothermia could complement each other in their specific mechanisms to provide neuroprotection.F.J.A. has a research agreement with GW Research Ltd, which is now a part of Jazz Pharmaceuticals, from which he receives financial support. W.H. is an employee Jazz Pharmaceuticals. The authors declare no other competing financial interests. The present study was supported by Grant GWCRI1547 (GW Research Ltd., now part of Jazz Pharmaceuticals); Grant PI12/0852 (ISCIII-General SubDirectorate for Research Assessment and Promotion and the European Regional Development Funds/European Social Fund: “A way to build Europe”); and Grant UPV GIU 17/18 (University of the Basque Country)

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Funding: This work was supported by CIBERobn (CB06/03/1008), Ministerio de Economía y Competitividad (MINECO) (PG: BFU2012-33334), Instituto de Salud Carlos III (ISCIII), MINECO, co-funded by UE-ERDF program (JS: CP12/03109), Red de Trastornos Adictivos (FRF: RD12/0028/0001, PG: RD12/0028/0004, JM: RD12/0028/0013), The Basque Country Government (PG: BCG IT764-13), Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, UE-ERDF (FRF: CTS-8221, JM: CVI-6656), Consejería de Salud, Junta de Andalucía, UE-ERDF (FRF: SAS111224), and University of the Basque Country UPV/EHU (PG: UFI11/41). JS, FJP and AS hold “Miguel Servet” research contracts from the National System of Health, ISCIII, UE-ERDF (CP12/03109, CP14/00212, and CP14/00173 respectively)Peer reviewedPublisher PD

GABAergic and Cortical and Subcortical Glutamatergic Axon Terminals Contain CB1 Cannabinoid Receptors in the Ventromedial Nucleus of the Hypothalamus

Background: Type-1 cannabinoid receptors (CB1R) are enriched in the hypothalamus, particularly in the ventromedial hypothalamic nucleus (VMH) that participates in homeostatic and behavioral functions including food intake. Although CB1R activation modulates excitatory and inhibitory synaptic transmission in the brain, CB1R contribution to the molecular architecture of the excitatory and inhibitory synaptic terminals in the VMH is not known. Therefore, the aim of this study was to investigate the precise subcellular distribution of CB1R in the VMH to better understand the modulation exerted by the endocannabinoid system on the complex brain circuitries converging into this nucleus. Methodology/Principal Findings: Light and electron microscopy techniques were used to analyze CB1R distribution in the VMH of CB1R-WT, CB1R-KO and conditional mutant mice bearing a selective deletion of CB1R in cortical glutamatergic (Glu-CB1R-KO) or GABAergic neurons (GABA-CB1R-KO). At light microscopy, CB1R immunolabeling was observed in the VMH of CB1R-WT and Glu-CB1R-KO animals, being remarkably reduced in GABA-CB1R-KO mice. In the electron microscope, CB1R appeared in membranes of both glutamatergic and GABAergic terminals/preterminals. There was no significant difference in the percentage of CB1R immunopositive profiles and CB1R density in terminals making asymmetric or symmetric synapses in CB1R-WT mice. Furthermore, the proportion of CB1R immunopositive terminals/preterminals in CB1R-WT and Glu-CB1R-KO mice was reduced in GABA-CB1R-KO mutants. CB1R density was similar in all animal conditions. Finally, the percentage of CB1R labeled boutons making asymmetric synapses slightly decreased in Glu-CB1R-KO mutants relative to CB1R-WT mice, indicating that CB1R was distributed in cortical and subcortical excitatory synaptic terminals. Conclusions/Significance: Our anatomical results support the idea that the VMH is a relevant hub candidate in the endocannabinoid-mediated modulation of the excitatory and inhibitory neurotransmission of cortical and subcortical pathways regulating essential hypothalamic functions for the individual's survival such as the feeding behavior.L. Reguero is in receipt of a Predoctoral Fellowship from the Basque Country Government (BFI 07.286); I. Buceta is in receipt of a Predoctoral Fellowship from the Basque Country University. Dr. Pedro Grandes' laboratory is supported by The Basque Country Government grant GIC07/70-IT-432-07, by Ministerio de Ciencia e Innovacion (SAF2009-07065) and by Red de Trastornos Adictivos, RETICS, Instituto de Salud Carlos III, MICINN, grant RD07/0001/2001. Dr. Giovanni Marsicano's laboratory is supported by AVENIR/INSERM (with the Fondation Bettencourt-Schueller), by ANR (ANR-06-NEURO-043-01), by European Foundation for the Study of Diabetes (EFSD), by the EU-FP7 (REPROBESITY, contract number HEALTH-F2-2008-223713) and European Commission Coordination Action ENINET (contract number LSHM-CT-2005-19063). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Visualization by high resolution immunoelectron microscopy of the transient receptor potential vanilloid-1 at inhibitory synapses of the mouse dentate gyrus.

We have recently shown that the transient receptor potential vanilloid type 1 (TRPV1), a non-selective cation channel in the peripheral and central nervous system, is localized at postsynaptic sites of the excitatory perforant path synapses in the hippocampal dentate molecular layer (ML). In the present work, we have studied the distribution of TRPV1 at inhibitory synapses in the ML. With this aim, a preembedding immunogold method for high resolution electron microscopy was applied to mouse hippocampus. About 30% of the inhibitory synapses in the ML are TRPV1 immunopositive, which is mostly localized perisynaptically (∼60% of total immunoparticles) at postsynaptic dendritic membranes receiving symmetric synapses in the inner 1/3 of the layer. This TRPV1 pattern distribution is not observed in the ML of TRPV1 knock-out mice. These findings extend the knowledge of the subcellular localization of TRPV1 to inhibitory synapses of the dentate molecular layer where the channel, in addition to excitatory synapses, is present

Subcellular distribution of TRPV1 at symmetric synapses in WT mouse dentate ML.

<p>Preembedding immunogold method for electron microscopy (<b>A-C</b>). TRPV1 immunoparticles are distributed in dendritic (den) sections receiving symmetric synapses (white arrowheads) from axon terminals (ter). Note that the metal particles (black arrowheads) are localized on the membranes and inside the dendritic profiles. Scale bars: 0.5 μm. <b>D:</b> Distribution of TRPV1 in dendrites (membrane: 75.25 ± 5.56%; inside: 24.75 ± 5.56%). Values mean ± SEM. (<i>***P<0</i>.<i>0001)</i>. <b>E:</b> Distribution of TRPV1 immunoparticles relative to the edge of postsynaptic membranes of symmetric synapses. The edge was defined as 0 with the peri/extrasynaptic side to the right. Note that the closest perisynaptic region (0–60 nm bin) contains the highest TRPV1 labeling (59.49%).</p

Expression and function of Kv7.4 channels in rat cardiac mitochondria:Possible targets for cardioprotection

AIMS: Plasmalemmal Kv7.1 (KCNQ1) channels are critical players in cardiac excitability; however, little is known on the functional role of additional Kv7 family members (Kv7.2-5) in cardiac cells. In this work, the expression, function, cellular and subcellular localization, and potential cardioprotective role against anoxic-ischaemic cardiac injury of Kv7.4 channels have been investigated. METHODS AND RESULTS: Expression of Kv7.1 and Kv7.4 transcripts was found in rat heart tissue by quantitative polymerase chain reaction. Western blots detected Kv7.4 subunits in mitochondria from Kv7.4-transfected cells, H9c2 cardiomyoblasts, freshly isolated adult cardiomyocytes, and whole hearts. Immunofluorescence experiments revealed that Kv7.4 subunits co-localized with mitochondrial markers in cardiac cells, with ∼30-40% of cardiac mitochondria being labelled by Kv7.4 antibodies, a result also confirmed by immunogold electron microscopy experiments. In isolated cardiac (but not liver) mitochondria, retigabine (1-30 µM) and flupirtine (30 µM), two selective Kv7 activators, increased Tl+ influx, depolarized the membrane potential, and inhibited calcium uptake; all these effects were antagonized by the Kv7 blocker XE991. In intact H9c2 cells, reducing Kv7.4 expression by RNA interference blunted retigabine-induced mitochondrial membrane depolarization; in these cells, retigabine decreased mitochondrial Ca2+ levels and increased radical oxygen species production, both effects prevented by XE991. Finally, retigabine reduced cellular damage in H9c2 cells exposed to anoxia/re-oxygenation and largely prevented the functional and morphological changes triggered by global ischaemia/reperfusion (I/R) in Langendorff-perfused rat hearts. CONCLUSION: Kv7.4 channels are present and functional in cardiac mitochondria; their activation exerts a significant cardioprotective role, making them potential therapeutic targets against I/R-induced cardiac injury

Immunolocalization of TRPV1 at symmetric synapses in mouse dentate ML.

<p>Preembedding immunogold method for electron microscopy (<b>A-C</b>). In TRPV1-WT (<b>A, B</b>), silver-intensified gold particles (arrowheads) are mostly distributed in dendritic (den) profiles but not in inhibitory synaptic terminals (ter). Importantly, TRPV1 immunolabeling is virtually abolished in the ML of TRPV1-KO mice (<b>C</b>). Scale bars: 0.5 μm. <b>D:</b> Percentages of TRPV1 positive dendritic profiles from the total analyzed dendritic sections with symmetric synapses (arrows in <b>A-C</b>) in the dentate ML of TRPV1-WT (dendrites: 32.89 ± 5.04%) and TRPV1-KO (dendrites: 4.76 ± 1.75%). Values in <b>D</b> mean ± SEM. <i>***P<0</i>.<i>0001</i>.</p

Immunolocalization of TRPV1 at dendrites in mouse dentate ML.

<p>Preembedding immunogold method for electron microscopy (<b>A-D</b>). TRPV1 immunolocalization (arrowheads) at dendritic (den) membranes receiving symmetric synapses (arrows) from axon terminals (ter) in the inner 1/3 (<b>A, B</b>) and outer 2/3 ML (<b>C, D</b>). Scale bars: 0.5 μm. <b>E:</b> Proportion of TRPV1 positive dendritic sections with symmetric synapses in the inner 1/3 (81.4%) and outer 2/3 (18.6%) of the dentate ML. <b>F:</b> Density of TRPV1 immunoparticles per area (part/μm²) in dendritic domains of the inner 1/3 (3.72 ± 0.85%) and outer 2/3 ML (1.95 ± 1.02%). Values mean ± SEM (<i>***P<0</i>.<i>0001</i>).</p

Immunolocalization of TRPV1 in granule cell perikarya of the mouse dentate ML.

<p>A high accumulation of TRPV1 immunoparticles is observed in the granule cytoplasm (cyt) of TRPV1-WT mouse (<b>A, B</b>). <b>A, B:</b> Arrows point to symmetric synapses made by axon terminals (ter) on the plasmalemma of granule cells. <b>C:</b> Notice the lack of TRPV1 labeling in the somatic cytoplasm of TRPV1-KO mice, but unspecific immunoparticles remain in the nucleus (nuc) that practically disappear after omission of the primary antibody (<b>D</b>). Scale bars: 0.5 μm. <b>E:</b> Density of TRPV1 immunoparticles per area (part/μm²) in the somatic cytoplasm of granule cells (TRPV1-WT 8.91 ± 0.58; TRPV1-KO 0.61 ± 0.11). Values mean ± SEM (<i>***P<0</i>.<i>0001</i>).</p