Marco Koch

2‐Arachidonoylglycerol elicits neuroprotective effects on excitotoxically lesioned dentate gyrus granule cells via abnormal‐cannabidiol‐sensitive receptors on microglial cells

Endocannabinoids like 2‐arachidonoylglycerol (2‐AG) exert neuroprotective effects after brain injuries. According to current concepts, these neuroprotective effects are due to interactions between 2‐AG and cannabinoid (CB)1 receptors on neurons. Moreover, 2‐AG modulates migration and proliferation of microglial cells which are rapidly activated after brain lesion. This effect is mediated via CB2‐ and abnormal‐cannabidiol (abn‐CBD)‐sensitive receptors. In the present study, we investigated whether the abn‐CBD‐sensitive receptor on microglial cells contributes to 2‐AG‐mediated neuroprotection in organotypic hippocampal slice cultures (OHSCs) after excitotoxic lesion induced by NMDA (50 μM) application for 4 h. This lesion caused neuronal damage and accumulation of microglial cells within the granule cell layer. To analyze the role of abn‐CBD‐sensitive receptors for neuroprotection and microglial cell accumulation, two agonists of the abn‐CBD‐sensitive receptor, abn‐CBD or 2‐AG, two antagonists, 1,3‐dimethoxy‐5‐methyl‐2‐[(1R,6R)‐3‐methyl‐6‐(1‐methylethenyl)‐2‐cyclohexen1‐yl]‐benzene (O‐1918) or cannabidiol (CBD), and the CB1 receptor antagonist AM251, were applied to NMDA‐lesioned OHSC. Propidium iodide (PI) labeling was used as a marker of degenerating neurons and isolectin B4 (IB4) as a marker of microglial cells. Application of both, abn‐CBD or 2‐AG to lesioned OHSC significantly decreased the number of IB  4+ microglial cells and PI+ neurons in the dentate gyrus. In contrast to AM251, application of O‐1918 or CBD antagonized these effects. When microglial cells were depleted by preincubation of OHSC with the bisphosphonate clodronate (100 μg/mL) for 5 days before excitotoxic lesion, 2‐AG and abn‐CBD lost their neuroprotective effects. We therefore propose that the endocannabinoid 2‐AG exerts its neuroprotective effects via activation of abn‐CBD‐sensitive receptors on microglial cells.

R-Flurbiprofen Reduces Neuropathic Pain in Rodents by Restoring Endogenous Cannabinoids

Background R-flurbiprofen, one of the enantiomers of flurbiprofen racemate, is inactive with respect to cyclooxygenase inhibition, but shows analgesic properties without relevant toxicity. Its mode of action is still unclear. Methodology/Principal Findings We show that R-flurbiprofen reduces glutamate release in the dorsal horn of the spinal cord evoked by sciatic nerve injury and thereby alleviates pain in sciatic nerve injury models of neuropathic pain in rats and mice. This is mediated by restoring the balance of endocannabinoids (eCB), which is disturbed following peripheral nerve injury in the DRGs, spinal cord and forebrain. The imbalance results from transcriptional adaptations of fatty acid amide hydrolase (FAAH) and NAPE-phospholipase D, i.e. the major enzymes involved in anandamide metabolism and synthesis, respectively. R-flurbiprofen inhibits FAAH activity and normalizes NAPE-PLD expression. As a consequence, R-Flurbiprofen improves endogenous cannabinoid mediated effects, indicated by the reduction of glutamate release, increased activity of the anti-inflammatory transcription factor PPARγ and attenuation of microglia activation. Antinociceptive effects are lost by combined inhibition of CB1 and CB2 receptors and partially abolished in CB1 receptor deficient mice. R-flurbiprofen does however not cause changes of core body temperature which is a typical indicator of central effects of cannabinoid-1 receptor agonists. Conclusion Our results suggest that R-flurbiprofen improves the endogenous mechanisms to regain stability after axonal injury and to fend off chronic neuropathic pain by modulating the endocannabinoid system and thus constitutes an attractive, novel therapeutic agent in the treatment of chronic, intractable pain.

Multiple ecto-nucleotidases in PC12 cells: identification and cellular distribution after heterologous expression.

The physiological action of extracellular ATP and other nucleotides in the nervous system is controlled by surface‐located enzymes (ecto‐nucleotidases) of which several families with partially overlapping substrate specificities exist. In order to identify ecto‐nucleotidases potentially associated with neural cells, we chose PC12 cells for analysis. PC12 cells revealed surface‐located ATPase and ADPase activity with apparent Km‐values of 283 µm and 243 µm, respectively. Using PCR we identified the mRNA of all members of the ecto‐nucleoside triphosphate diphosphohydrolase family investigated (NTPDase1 to NTPDase3, NTPDase5/6), of ecto‐nucleotide pyrophosphatase/phosphodiesterase3 (NPP3), tissue‐non‐specific alkaline phosphatase and ecto‐5′‐nucleotidase. The surface‐located catalytic activity differed greatly between the various enzyme species. Our data suggest that hydrolysis of ATP and ADP is mainly due to members of the ecto‐nucleoside triphosphate diphosphohydrolase family. Activity of ecto‐5′‐nucleotidase and alkaline phosphatase was very low and activity of NPP3 was absent. For a detailed analysis of the cellular distribution of ecto‐nucleotidases single and double transfections of PC12 cells were performed, followed by fluorescence analysis. Ecto‐nucleotidases were distributed over the entire cell surface and accumulated intracellularly in varicosities and neurite tips. PC12 cell ecto‐nucleotidases are likely to play an important role in terminating autocrine functions of released nucleotides and in producing extracellular nucleosides supporting the survival and neuritic differentiation of PC12 cells.

Cannabinoids and neuronal damage: Differential effects of THC, AEA and 2-AG on activated microglial cells and degenerating neurons in excitotoxically lesioned rat organotypic hippocampal slice cultures

Cannabinoids (CBs) are attributed neuroprotective effects in vivo. Here, we determined the neuroprotective potential of CBs during neuronal damage in excitotoxically lesioned organotypic hippocampal slice cultures (OHSCs). OHSCs are the best characterized in vitro model to investigate the function of microglial cells in neuronal damage since blood-borne monocytes and T-lymphocytes are absent and microglial cells represent the only immunocompetent cell type. Excitotoxic neuronal damage was induced by NMDA (50 microM) application for 4 h. Neuroprotective properties of 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), N-arachidonoylethanolamide (AEA) or 2-arachidonoylglycerol (2-AG) in different concentrations were determined after co-application with NMDA by counting degenerating neurons identified by propidium iodide labeling (PI(+)) and microglial cells labeled by isolectin B(4) (IB(4)(+)). All three CBs used significantly decreased the number of IB(4)(+) microglial cells in the dentate gyrus but the number of PI(+) neurons was reduced only after 2-AG treatment. Application of AM630, antagonizing CB2 receptors highly expressed by activated microglial cells, did not counteract neuroprotective effects of 2-AG, but affected THC-mediated reduction of IB(4)(+) microglial cells. Our results indicate that (1) only 2-AG exerts neuroprotective effects in OHSCs; (2) reduction of IB(4)(+) microglial cells is not a neuroprotective event per se and involves other CB receptors than the CB2 receptor; (3) the discrepancy in the neuroprotective effects of CBs observed in vivo and in our in vitro model system may underline the functional relevance of invading monocytes and T-lymphocytes that are absent in OHSCs.

Dephosphorylation of pCREB by protein serine/threonine phosphatases is involved in inactivation of Aanat gene transcription in rat pineal gland

The rat pineal gland is a suitable model to investigate neurotransmitter‐controlled gene expression, because it is well established that the stimulation of melatonin biosynthesis by norepinephrine (NE) depends on the activation of the gene that encodes arylalkylamine N‐acetyltransferase (AANAT), the melatonin rhythm enzyme. The mechanisms responsible for downregulation of Aanat transcription are less clear. In this in vitro study we investigated the role of pCREB dephosphorylation for termination of Aanat gene transcription. Immunosignals for pCREB, strongly induced after NE stimulation, rapidly decreased after withdrawal of NE. The immunoreactivity of the inhibitory transcription factor ICER increased twofold after NE treatment for 6 h, but did not change within 30 min after removal of the stimulus. Application of protein serine/threonine phosphatase (PSP) inhibitors prevented pCREB dephosphorylation and blocked the decreases in Aanat mRNA levels, AANAT protein amount and melatonin biosynthesis all of which occurred rapidly after NE withdrawal. PSPs in the rat pineal gland were characterized by immunocytochemistry and immunoblotting. NE‐stimulation for 8 h induced accumulation of PSP1‐catalytic subunit (CSU) in pinealocyte nuclei, but did not affect the distribution of PSP2A‐CSU. The results identify dephosphorylation of pCREB by PSPs as an essential mechanism for downregulation of Aanat transcription in the rat pineal gland.

The G Protein‐Coupled Receptor 55 Ligand l‐α‐Lysophosphatidylinositol Exerts Microglia‐Dependent Neuroprotection After Excitotoxic Lesion

Searching for chemical agents and molecular targets protecting against secondary neuronal damage reflects one major issue in neuroscience. Cannabinoids limit neurodegeneration by activation of neuronal G protein‐coupled cannabinoid receptor 1 (CB1) and microglial G protein‐coupled cannabinoid receptor 2 (CB2). However, pharmacological experiments with CB1/CB2‐deficient mice unraveled the existence of further, so‐called non‐CB1/non‐CB2 G protein‐coupled receptor (GPR) subtypes. GPR55, whose function in the brain is still poorly understood, represents a novel target for various cannabinoids. Here, we investigated whether GPR55 reflects a potential beneficial target in neurodegeneration by using the excitotoxicity in vitro model of rat organotypic hippocampal slice cultures (OHSC). l‐α‐Lysophosphatidylinositol (LPI), so far representing the most selective agonist for GPR55, protected dentate gyrus granule cells and reduced the number of activated microglia after NMDA (50 µM) induced lesions. The relevance of GPR55 activation for LPI‐mediated neuroprotection was determined by using Gpr55 siRNA. Microglia seems to mediate the observed neuroprotection since their depletion in OHSC attenuated the beneficial effects of LPI. Moreover, LPI alone induced microglia chemotaxis but conversely significantly attenuated ATP triggered microglia migration. These effects seemed to be independent from intracellular Ca2+ and p38 or p44/p42 MAPK phosphorylation. In conclusion, this study unmasked a yet unknown role for GPR55 in neuroprotection driven by LPI‐mediated modulation of microglia function. GLIA 2013;61:1822–1831

An endocannabinoid system is localized to the hypophysial pars tuberalis of Syrian hamsters and responds to photoperiodic changes.

The hypophysial pars tuberalis (PT), an important interface between neuroendocrine brain centers (hypothalamus, pineal organ) and the pars distalis (PD) of the hypophysis, plays a central role in regulating seasonal reproduction and prolactin release. However, the signaling molecules that transmit photoperiodic information from the PT to the PD and control prolactin release (the so-called “tuberalins”) have not yet been identified, despite an intense search for more than three decades. Here, we demonstrate an endocannabinoid system in the PT of the Syrian hamster, a photoperiodic species. By means of in situ hybrization, the PT was found to express N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH), sn-1-selective diacylglycerol lipases (DAGLα and DAGLβ), and monoacylglycerol lipase (MAGL), enzymes involved in endocannabinoid synthesis and degradation. The expression of NAPE-PLD, FAAH, and DAGLα was confirmed by immunohistochemistry. Expression and protein levels of DAGLs controlling the synthesis of 2-arachidonoyl glycerol (2-AG), a major endocannabinoid, were upregulated in the PT of Syrian hamsters kept under long-day conditions. Consequently, 2-AG levels were increased in the PT of these hamsters. A primary target of 2-AG, the cannabinoid receptor 1 (CB1), was expressed in the PD. Double-immunolabeling revealed that most of the CB1-immunoreactive cells in the PD were folliculostellate cells that were also immunoreactive for S-100 protein. Thus, the PT comprises an endocannabinoid system, and 2-AG may act as a photoperiodic messenger from the PT to the PD for the regulation of hypophysial hormonal secretion.

The cannabinoid WIN 55,212-2-mediated protection of dentate gyrus granule cells is driven by CB1 receptors and modulated by TRPA1 and Cav2.2 channels

Cannabinoids regulate numerous physiological and pathological events like inflammation or neurodegeneration via CB1 and CB2 receptors. The mechanisms behind cannabinoid effects show a high variability and may also involve transient receptor potential channels (TRP) and N‐type voltage‐gated Ca2+ channels (Cav2.2). In the present study we investigated the neuroprotective effects of the synthetic cannabinoid WIN 55,212–2 (WIN) on dentate gyrus (DG) granule cells and elucidated the involvement of TRP and Cav2.2 that are shown to participate in inflammatory processes. Organotypic hippocampal slice cultures were excitotoxically lesioned using NMDA and subsequently incubated with different WIN concentrations (0.001–10 μM). WIN showed neuroprotective properties in an inverse concentration‐dependent manner, most effectively at 0.01 μM. The CB1 receptor antagonist AM251 blocked neuroprotection mediated by WIN whereas the CB2 receptor antagonist AM630 showed no effects. Application of the TRPA1 blocker HC‐030031 enhanced the neuroprotective efficacy of high (10 μM) WIN concentrations and the number of degenerating neurons became equal to that seen after application of the most effective WIN dose (0.01 μM). In contrast, the application of TRPA1 agonist icilin or allyl isothiocyanate (AITC) led to a stronger neurodegeneration. The use of TRPV1 blocker 6‐iodo‐nordihydrocapsaicin did not affect WIN‐mediated neuroprotection. The selective Cav2.2 blocker ω‐conotoxin (GVIA) completely blocked neuroprotection shown by 10 μM WIN. GVIA and HC‐030031 exerted no effects at WIN concentrations lower than 10 μM. Our data show that WIN protects dentate gyrus granule cells in a concentration dependent manner by acting upon CB1 receptors. At high (10 μM) concentrations WIN additionally activates TRPA1 and Cav2.2 within the hippocampal formation that both interfere with CB1 receptor‐mediated neuroprotection. This leads to the conclusion that physiological and pharmacological effects of cannabinoids strongly depend on their concentration and the neuroprotective efficacy of cannabinoids may be determined by interaction of activated CB1 receptor, TRPA1, and Cav2.2.

Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain.

Immunohistochemical visualization of antigens in specimen has evolved to an indispensable technique in biomedical research for investigations of cell morphology and pathology both in bright field and fluorescence microscopy. While there are couple of staining methods that reveal entire cytoarchitecture in bright field microscopy such as Nissl or hemalaun-eosin, there are still limitations in visualizations of cytoarchitecture in fluorescence microscopy. The present study reports a simple staining method that provides the required illustration of cell allocations and cellular composition in fluorescence microscopy in adult and in developing rodent central nervous system using the fluorophore propidium iodide (PI, 5μg/mL). PI is a well-accepted marker for degenerating cells when applied prior to fixation (pre-fixation PI staining). Here, PI was added to the sections after the fixation (post-fixation PI staining). This revised labeling procedure led to similar cytoarchitectural staining patterns in fluorescence microscopy as observed with hemalaun in bright field microscopy. This finding was proven in organotypic hippocampal slice cultures (OHSC) and brain sections obtained from different postnatal developmental stages. Excitotoxically lesioned OHSC subjected to pre-fixation PI staining merely showed brightly labeled condensed nuclei of degenerating neurons. In contrast, post-fixation PI staining additionally revealed extensive labeling of neuronal cell bodies and glial cells within the OHSC, thus allowing visualization of stratification of neuronal layers and cell morphology. Furthermore, post-fixation PI staining was combined with NeuN, calbindin, calretinin, glial fibrillary acidic protein or Griffonia simplicifolia isolectin B4 (IB(4)) in post natal (p1 and p9) and adult rats. In early post-natal brain sections almost all mentioned cellular markers led to an incomplete staining of the native cell organization and resulted in an inaccurate estimation of cell morphology when compared to adult brains. In contrast, post-fixation PI staining allowed investigation of the whole cytoarchitecture independent of the developmental stage. Taken together, post-fixation PI staining provides a detailed insight in the morphology of both developing and adult brain tissues in fluorescence microscopy.

The rat pineal gland comprises an endocannabinoid system.

Abstract:  In the mammalian pineal gland, the rhythm in melatonin biosynthesis depends on the norepinephrine (NE)‐driven regulation of arylalkylamine N‐acetyltransferase (AANAT), the penultimate enzyme of melatonin biosynthesis. A recent study showed that phytocannabinoids like tetrahydrocannabinol reduce AANAT activity and attenuate NE‐induced melatonin biosynthesis in rat pineal glands, raising the possibility that an endocannabinoid system is present in the pineal gland. To test this hypothesis, we analyzed cannabinoid (CB) receptors and specific enzymes for endocannabinoid biosynthesis or catabolism in rat pineal glands and cultured pinealocytes. Immunohistochemical and immunoblot analyses revealed the presence of CB1 and CB2 receptor proteins, of N‐acyl phosphatidyl ethanolamine hydrolyzing phospholipase D (NAPE‐PLD), an enzyme catalyzing endocannabinoid biosynthesis and of fatty acid amide hydrolase (FAAH), an endocannabinoid catabolizing enzyme, in pinealocytes, and in pineal sympathetic nerve fibers identified by double immunofluorescence with an antibody against tyrosine hydroxylase. The immunosignals for the CB2 receptor, NAPE‐PLD, and FAAH found in pinealocytes did not vary under a 12 hr light:12 hr dark cycle. The CB1 receptor immunoreaction in pinealocytes was significantly reduced at the end of the light phase [zeitgeber time (ZT) 12]. The immunosignal for NAPE‐PLD found in pineal sympathetic nerve fibers was reduced in the middle of the dark phase (ZT 18). Stimulation of cultured pinealocytes with NE affected neither the subcellular distribution nor the intensity of the immunosignals for the investigated CB receptors and enzymes. In summary, the pineal gland comprises indispensable compounds of the endocannabinoid system indicating that endocannabinoids may be involved in the control of pineal physiology.