Accumulating evidence supports the role of astrocytes in endocannabinoid mediated modulation of neural activity. It has been reported that some astrocytes express the cannabinoid type 1 receptor (CB1-R), the activation of which is leading to Ca2+ mobilization from internal stores and a consecutive release of glutamate. It has also been documented that astrocytes have the potential to produce the endocannabinoid 2-arachidonoylglycerol, one of the best known CB1-R agonist. However, no relationship between CB1-R activation and 2-arachidonoylglycerol production has ever been demonstrated. Here we show that rat spinal astrocytes co-express CB1-Rs and the 2-arachidonoylglycerol synthesizing enzyme, diacylglycerol lipase-alpha in close vicinity to each other. We also demonstrate that activation of CB1-Rs induces a substantial elevation of intracellular Ca2+ concentration in astrocytes. Finally, we provide evidence that the evoked Ca2+ transients lead to the production of 2-arachidonoylglycerol in cultured astrocytes. The results provide evidence for a novel cannabinoid induced endocannabinoid release mechanism in astrocytes which broadens the bidirectional signaling repertoire between astrocytes and neurons.
The pedunculopontine nucleus (PPN) is known as the cholinergic part of the reticular activating system (RAS) and it plays an important role in transitions of slow-wave sleep to REM sleep and wakefulness. Although both exogenous and endocannabinoids affect sleep, the mechanism of endocannabinoid neuromodulation has not been characterized at cellular level in the PPN. In this paper, we demonstrate that both neurons and glial cells from the PPN respond to cannabinoid type 1 (CB1) receptor agonists. The neuronal response can be depolarization or hyperpolarization, while astrocytes exhibit more frequent calcium waves. All these effects are absent in CB1 gene-deficient mice. Blockade of the fast synaptic neurotransmission or neuronal action potential firing does not change the effect on the neuronal membrane potential significantly, while inhibition of astrocytic calcium waves by thapsigargin diminishes the response. Inhibition of group I metabotropic glutamate receptors (mGluRs) abolishes hyperpolarization, whereas blockade of group II mGluRs prevents depolarization. Initially active neurons and glial cells display weaker responses partially due to the increased endocannabinoid tone in their environment. Taken together, we propose that cannabinoid receptor stimulation modulates PPN neuronal activity in the following manner: active neurons may elicit calcium waves in astrocytes via endogenous CB1 receptor agonists. Astrocytes in turn release glutamate that activates different metabotropic glutamate receptors of neurons and modulate PPN neuronal activity.
The orbitofrontal cortex (OFC) has been implicated in a multiplicity of complex brain functions, including representations of expected outcome properties, post-decision confidence, momentary food-reward values, complex flavors and odors. As breathing rhythm has an influence on odor processing at primary olfactory areas, we tested the hypothesis that it may also influence neuronal activity in the OFC, a prefrontal area involved also in higher order processing of odors. We recorded spike timing of orbitofrontal neurons as well as local field potentials (LFPs) in awake, head-fixed mice, together with the breathing rhythm. We observed that a large majority of orbitofrontal neurons showed robust phase-coupling to breathing during immobility and running. The phase coupling of action potentials to breathing was significantly stronger in orbitofrontal neurons compared to cells in the medial prefrontal cortex. The characteristic synchronization of orbitofrontal neurons with breathing might provide a temporal framework for multi-variable processing of olfactory, gustatory and reward-value relationships.
Protein phosphatase‐1M (PP1M, myosin phosphatase) consists of a PP1 catalytic subunit (PP1c) and the myosin phosphatase target subunit‐1 (MYPT1). RhoA‐activated kinase (ROK) regulates PP1M via inhibitory phosphorylation of MYPT1. Using multidisciplinary approaches, we have studied the roles of PP1M and ROK in neurotransmission. Electron microscopy demonstrated the presence of MYPT1 and ROK in both pre‐ and post‐synaptic terminals. Tautomycetin (TMC), a PP1‐specific inhibitor, decreased the depolarization‐induced exocytosis from cortical synaptosomes. trans‐4‐[(1R)‐1‐aminoethyl]‐N‐4‐pyridinylcyclohexanecarboxamide dihydrochloride, a ROK‐specific inhibitor, had the opposite effect. Mass spectrometry analysis identified several MYPT1‐bound synaptosomal proteins, of which interactions of synapsin‐I, syntaxin‐1, calcineurin‐A subunit, and Ca2+/calmodulin‐dependent kinase II with MYPT1 were confirmed. In intact synaptosomes, TMC increased, whereas Y27632 decreased the phosphorylation levels of MYPT1Thr696, myosin‐II light chainSer19, synapsin‐ISer9, and syntaxin‐1Ser14, indicating that PP1M and ROK influence their phosphorylation status. Confocal microscopy indicated that MYPT1 and ROK are present in the rat ventral cochlear nucleus both pre‐ and post‐synaptically. Analysis of the neurotransmission in an auditory glutamatergic giant synapse demonstrated that PP1M and ROK affect neurotransmission via both pre‐ and post‐synaptic mechanisms. Our data suggest that both PP1M and ROK influence synaptic transmission, but further studies are needed to give a full account of their mechanism of action.
Retinoids are morphogens and have been implicated in cell fate commitment of embryonic stem cells (ESCs) to neurons. Their effects are mediated by RAR and RXR nuclear receptors. However, transcriptional cofactors required for cell and gene-specific retinoid signaling are not known. Here we show that protein arginine methyl transferase (PRMT) 1 and 8 have key roles in determining retinoid regulated gene expression and cellular specification in a multistage neuronal differentiation model of murine ESCs. PRMT1 acts as a selective modulator, providing the cells with a mechanism to reduce the potency of retinoid signals on regulatory "hotspots." PRMT8 is a retinoid receptor target gene itself and acts as a cell type specific transcriptional coactivator of retinoid signaling at later stages of differentiation. Lack of either of them leads to reduced nuclear arginine methylation, dysregulated neuronal gene expression, and altered neuronal activity. Importantly, depletion of PRMT8 results in altered expression of a distinct set of genes, including markers of gliomagenesis. PRMT8 is almost entirely absent in human glioblastoma tissues. We propose that PRMT1 and PRMT8 serve as a rheostat of retinoid signaling to determine neuronal cell specification in a context-dependent manner and might also be relevant in the development of human brain malignancy. STEM CELLS 2015;33:726-741
Although cholinergic modulation of the cochlear nucleus (CN) is functionally important, neither its cellular consequences nor the types of receptors conveying it are precisely known. The aim of this work was to characterise the cholinergic effects on giant cells of the CN, using electrophysiology and quantitative polymerase chain reaction. Application of the cholinergic agonist carbachol increased the spontaneous activity of the giant cells; which was partly the consequence of the reduction in a K(+) conductance. This effect was mediated via M4 and M3 receptors. Cholinergic modulation also affected the synaptic transmission targeting the giant cells. Excitatory synaptic currents evoked by the stimulation of the superficial and deep regions of the CN were sensitive to cholinergic modulation: the amplitude of the first postsynaptic current was reduced, and the short-term depression was also altered. These changes were mediated via M3 receptors alone and via the combination of M4, M2 and M3 receptors, when the superficial and deep layers, respectively, were activated. Inhibitory synaptic currents evoked from the superficial layer showed short-term depression, but they were unaffected by carbachol. In contrast, inhibitory currents triggered by the activation of the deep parts exhibited no significant short-term depression, but they were highly sensitive to cholinergic activation, which was mediated via M3 receptors. Our results indicate that pre- and postsynaptic muscarinic receptors mediate cholinergic modulation on giant cells. The present findings shed light on the cellular mechanisms of a tonic cholinergic modulation in the CN, which may become particularly important in evoking contralateral excitatory responses under certain pathological conditions.
Acetylcholine modulates the function of the cochlear nucleus via several pathways. In this study the effects of cholinergic stimulation were studied on the cytoplasmic Ca 2+ concentration of granule neurones of the rat dorsal cochlear nucleus (DCN). Ca 2+ transients were recorded in Oregon-Green-BAPTA 1-loaded brain slices using a calcium imaging technique. For the detection, identification, and characterisation of the Ca 2+ transients, a wavelet analysis-based method was developed. Granule cells were identified on the basis of their size and localisation. The action potential-coupled character of the Ca 2+ transients of the granule cells was established by recording fluorescence changes and electrical activity simultaneously. Application of the cholinergic agonist carbamyl-choline (CCh) significantly increased the frequency of the Ca 2+ transients (from 0.37 to 6.31 min -1 , corresponding to a 17.1-fold increase; n = 89).This effect was antagonised by atropine, whereas CCh could still evoke an 8.3-fold increase of the frequency of the Ca 2+ transients when hexamethonium was present.Using immunolabelling, the expression of both type 1 and type 3 muscarinic receptors (M1 and M3 receptors, respectively) was demonstrated in the granule cells. Application of 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (an M3-specific antagonist) prevented the onset of the CCh effect, whereas an M1-specific antagonist (pirenzepine) was less effective. We conclude that cholinergic stimulation increases the activity of granule cells, mainly by acting on their M3 receptors. The modulation of the firing activity of the granule cells, in turn, may modify the firing of projection neurones, and may adjust signal processing in the entire DCN.
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