Localization of diacylglycerol lipase-alpha around postsynaptic spine suggests close proximity between production site of an endocannabinoid, 2-arachidonoyl-glycerol, and presynaptic cannabinoid CB1 receptor - PubMed (original) (raw)

Comparative Study

Localization of diacylglycerol lipase-alpha around postsynaptic spine suggests close proximity between production site of an endocannabinoid, 2-arachidonoyl-glycerol, and presynaptic cannabinoid CB1 receptor

Takayuki Yoshida et al. J Neurosci. 2006.

Abstract

2-arachidonoyl-glycerol (2-AG) is an endocannabinoid that is released from postsynaptic neurons, acts retrogradely on presynaptic cannabinoid receptor CB1, and induces short- and long-term suppression of transmitter release. To understand the mechanisms of the 2-AG-mediated retrograde modulation, we investigated subcellular localization of a major 2-AG biosynthetic enzyme, diacylglycerol lipase-alpha (DAGLalpha), by using immunofluorescence and immunoelectron microscopy in the mouse brain. In the cerebellum, DAGLalpha was predominantly expressed in Purkinje cells. DAGLalpha was detected on the dendritic surface and occasionally on the somatic surface, with a distal-to-proximal gradient from spiny branchlets toward somata. DAGLalpha was highly concentrated at the base of spine neck and also accumulated with much lower density on somatodendritic membrane around the spine neck. However, DAGLalpha was excluded from the main body of spine neck and head. In hippocampal pyramidal cells, DAGLalpha was also accumulated in spines. In contrast to the distribution in Purkinje cells, DAGLalpha was distributed in the spine head, neck, or both, whereas somatodendritic membrane was labeled very weakly. These results indicate that DAGLalpha is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two neurons. The preferential spine targeting should enable efficient 2-AG production on excitatory synaptic activity and its swift retrograde modulation onto nearby presynaptic terminals expressing CB1. Furthermore, different fine localization within and around spines suggests that the distance between postsynaptic 2-AG production site and presynaptic CB1 is differentially controlled depending on neuron types.

PubMed Disclaimer

Figures

Figure 1.

Specificity of DAGLα antibody and immunohistochemistry. A, Immunoblot. Rabbit (Rb) and guinea pig (GP) antibodies to DAGLα recognize a 105 or 120 kDa protein band in the hippocampus (Hi) and cerebellum (Cb), respectively. The position of standard protein markers is indicated to the left (kDa). B, Immunofluorescence for DAGLα in the adult mouse brain. Inset shows blank immunostaining in a control experiment using antibody preabsorbed with antigen. C–F, In situ hybridization for DAGLα (C–E) and DAGLβ (F) mRNAs in the adult mouse brain. CP, Caudate–putamen; Cx, cerebral cortex; GL, granular layer; Mb, midbrain; ML, molecular layer; MO, medulla oblongata; OB, olfactory bulb; PC, Purkinje cell layer; Py, pyramidal cell layer; Ra, stratum radiatum; Th, thalamus. Scale bars: B, C, F, 1 mm; D, E, 10 μm.

Figure 2.

Immunofluorescence showing Purkinje cell-specific and somatodendritic distribution of DAGLα in the cerebellum. A–D, Double immunofluorescence for DAGLα (red) and calbindin (green). E, Triple immunofluorescence for DAGLα (red), parvalbumin (green), and calbindin (blue). F, Double immunofluorescence for DAGLα (red) and 3PGDH (green). A, Predominant distribution of DAGLα in Purkinje cell dendrites in the molecular layer (ML). Inset in A1 is an enlarged view showing stronger immunolabeling in spiny branchlets (SB) than in thick proximal dendrites (PD). Asterisks indicate Purkinje cell somata. B, Lack of DAGLα in dendritic spines. C, Occasional labeling of DAGLα in Purkinje cell soma (PC, arrowheads). D, Lack of DAGLα in Purkinje cell terminals on neurons in the deep cerebellar nuclei (DN). E, Lack of DAGLα in molecular layer interneurons (In). E2 is an image in which the light blue color is subtracted from E1. F, Lack of DAGLα in Bergmann glia. GL, Granular layer. Scale bars, 10 μm.

Figure 3.

Immunofluorescence showing anatomical relationship of DAGLα distribution with presynaptic and postsynaptic molecules. In all images, DAGLα is in red. A–C, Lack of DAGLα in VGluT2-labeled climbing fiber terminals (A), VGluT1-labeled parallel fiber terminals (B), and VGAT-labeled inhibitory interneuron terminals (C). D–F, DAGLα on spiny branchlets (SB) is distributed closely to but separated from CB1 (green in D; blue in F) or mGluR1α (green in E, F). G, Triple immunostaining for DAGLα, PLCβ4 (green), and mGluR1α (blue). Note overlap of PLCβ4 with DAGLα in dendritic shafts and with mGluR1α in dendritic spines. Scale bars: A–F, 10 μm; G, 5 μm; insets in D–F, 2 μm.

Figure 4.

Preembedding immunoelectron microscopy showing selective somatodendritic surface expression of DAGLα in Purkinje cells. A–D, Immunoperoxidase. E–G, Silver-enhanced immunogold. A, Spiny branchlet (SB). Note strong labeling on the surface of spiny branchlets but not in most portions of spines (Sp) contacting to parallel fiber terminals (PF). B, Proximal dendrite (PD). Note the lack of DAGLα at synaptic junction with inhibitory terminals (In). C, D, Purkinje cell body (PCB). DAGLα labeling is seen at the base of somatic spine contacting with a climbing fiber terminal (CF) and also on the somatic cell membrane around the spine. D is an enlarged view of the boxed region in C. E, Spiny branchlet (SB). Note the dense immunogold labeling on spiny branchlets but not on most portions of spine contacting with a parallel fiber terminal (PF). F, Proximal dendrite (PD). Immunogold labeling on proximal dendrites but not on most portions of spines contacting with a climbing fiber terminal (CF). G, Histograms showing the density of immunogold labeling for DAGLα on each domain of Purkinje cells. The total length of measured cell membranes is 48.5 μm in the soma, 103.3 μm in proximal dendrites, 76.3 μm in spiny branchlets, and 39.9 μm in spines. Arrows and arrowheads indicate the border between spines and dendrites (or soma) or the edge of postsynaptic density, respectively. Scale bars, 0.5 μm.

Figure 5.

Postembedding immunogold for DAGLα in dendritic spines of Purkinje cells. A, B, Two longitudinally sectioned parallel fiber synapses contacting to spines (Sp) emitting from spiny branchlets (SB). Note the prominent accumulation of immunogold labeling at the base of the spine neck. Also note the lack of immunogold labeling on most other parts of spines, including the spine head. PF, Parallel fiber. C, Histograms showing the distribution of gold particles within and around spines. The distance from the dendrite–spine border (0 nm in C; arrows in A, B) is plotted in the abscissa. The ordinate indicates the number of gold particles in each 100 nm bin, being summed up from 57 spines analyzed (n = 415 immunogold particles). D, Histograms showing the perpendicular distribution of DAGLα. The number of gold particles is plotted as a function of the distance from the midpoint of the plasma membrane to the center of gold particles (n = 471 immunogold particles). Arrowheads indicate the edge of postsynaptic density. Scale bars, 0.5 μm.

Figure 6.

Localization of CB1 at parallel fiber–Purkinje cell synapses by silver-enhanced immunogold. A, Double-immunoelectron microscopy by immunoperoxidase for DAGLα and by silver-enhanced immunogold for CB1 (open arrowheads). Arrows indicate the dendrite–spine border. B, Distribution of CB1 (open arrowheads) in a longitudinally sectioned parallel fiber (PF) synapse. Note the higher labeling density on the axolemma-facing spine (Sp) than that of the opposite-facing side. SB, Spiny branchlet. Scale bars, 0.5 μm.

Figure 7.

Spine- or synapse-associated expression of DAGLα in Purkinje cells at P10 (A–C) and adult (D, E). Triple labeling for DAGLα (red), VGluT2 (green), and calbindin (blue). Portions of A and D are enlarged in B and C or in E, respectively. Note that DAGLα is localized just beneath calbindin-stained somatic spines (arrowheads) and VGluT2-stained climbing fiber terminals (arrowheads) at both ages (B, E). Also note that distalmost dendrites in the superficial molecular layer at P10 are labeled less strongly for DAGLα than proximal shaft dendrites (C). EGL, External granular layer; GL, granular layer; ML, molecular layer; PC, Purkinje cell layer. Scale bars, 10 μm.

Figure 8.

Immunofluorescence for DAGLα in the hippocampal CA1 region. A, B, Double immunostaining for DAGLα (red) and the dendritic marker MAP2 (green). Note tiny punctate labeling for DAGLα around MAP2-positive thin dendrites. C, Triple immunostaining for DAGLα (red), VGAT (green), and VGluT1 (blue). D, Double immunostaining for DAGLα (red) and CB1 (green). LM, Stratum lacunosum-moleculare; Py, pyramidal cell; Ra, stratum radiatum. Scale bars, 10 μm.

Figure 9.

Immunoperoxidase (A–C) and silver-enhanced immunogold (D, E) electron microscopies for DAGLα in the hippocampal CA1 region. A–C, DAGLα is concentrated in dendritic spines of pyramidal cells but not in dendritic shafts (PyD). Immunoreaction products are seen in the spine neck (A), head (B), or both (C). D, E, Consecutive sections showing DAGLα labeling in the neck (D) and head (E) of spines. Arrowheads and arrows indicate the edge of postsynaptic density or the dendrite–spine border, respectively. F, Histograms showing the density of preembedding immunogold labeling for DAGLα on each domain of CA1 pyramidal cells. The total length of measured cell membranes is 24.4 μm in the soma, 31.4 μm in shaft dendrites, 225.5 μm in thin dendrites, and 120.5 μm in spines. G, Histograms showing the distribution of preembedding immunogold labeling in spines of CA1 pyramidal cells. The relative distance from the dendrite–spine border (0%) to the edge of the postsynaptic density (100%) is plotted in the abscissa. The ordinate indicates the percentage of metal particles in each bin, being summed from 49 spines analyzed (n = 76 particles). Arrows and arrowheads indicate the border between spines and dendrites (or soma) or the edge of the postsynaptic density, respectively. Scale bars, 0.5 μm.

Figure 10.

A scheme for endocannabinoid signaling at spiny branchlets of Purkinje cells (A) and hippocampal CA1 pyramidal cells (B), as deduced from molecular localization reported previously and in the present study. Ex, Excitatory terminal; In, inhibitory terminal; CCK-In, cholecystokinin-positive inhibitory terminal; PV-In, parvalbumin-positive inhibitory terminal; PCD, Purkinje cell dendrite; PF, parallel fiber terminal; PyD, pyramidal cell dendrite.

Cited by

Imaging and Genetic Tools for the Investigation of the Endocannabinoid System in the CNS.
Kouchaeknejad A, Van Der Walt G, De Donato MH, Puighermanal E. Kouchaeknejad A, et al. Int J Mol Sci. 2023 Oct 31;24(21):15829. doi: 10.3390/ijms242115829. Int J Mol Sci. 2023. PMID: 37958825 Free PMC article. Review.
Inhibiting degradation of 2-arachidonoylglycerol as a therapeutic strategy for neurodegenerative diseases.
Chen C. Chen C. Pharmacol Ther. 2023 Apr;244:108394. doi: 10.1016/j.pharmthera.2023.108394. Epub 2023 Mar 24. Pharmacol Ther. 2023. PMID: 36966972 Free PMC article. Review.
Endocannabinoid 2-Arachidonoylglycerol Synthesis and Metabolism at Neuronal Nuclear Matrix Fractions Derived from Adult Rat Brain Cortex.
Aretxabala X, García Del Caño G, Barrondo S, López de Jesús M, González-Burguera I, Saumell-Esnaola M, Goicolea MA, Sallés J. Aretxabala X, et al. Int J Mol Sci. 2023 Feb 5;24(4):3165. doi: 10.3390/ijms24043165. Int J Mol Sci. 2023. PMID: 36834575 Free PMC article.
Ventral hippocampal diacylglycerol lipase-alpha deletion decreases avoidance behaviors and alters excitation-inhibition balance.
Kondev V, Bluett R, Najeed M, Rosas-Vidal LE, Grueter BA, Patel S. Kondev V, et al. Neurobiol Stress. 2022 Dec 20;22:100510. doi: 10.1016/j.ynstr.2022.100510. eCollection 2023 Jan. Neurobiol Stress. 2022. PMID: 36594052 Free PMC article.
Regulation of adult neurogenesis by the endocannabinoid-producing enzyme diacylglycerol lipase alpha (DAGLa).
Schuele LL, Schuermann B, Bilkei-Gorzo A, Gorgzadeh S, Zimmer A, Leidmaa E. Schuele LL, et al. Sci Rep. 2022 Jan 12;12(1):633. doi: 10.1038/s41598-021-04600-1. Sci Rep. 2022. PMID: 35022487 Free PMC article.

References

1. Alger BE (2002). Retrograde signaling in the regulation of synaptic transmission: focus on endocannabinoids. Prog Neurobiol 68:247–286. - PubMed
1. Altman J (1972). Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. J Comp Neurol 145:399–463. - PubMed
1. Baude A, Nusser Z, Roberts JD, Mulvihill E, McIlhinney RA, Somogyi P (1993). The metabotropic glutamate receptor (mGluR1α) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction. Neuron 11:771–787. - PubMed
1. Baude A, Nusser Z, Molnar E, McIlhinney RA, Somogyi P (1995). High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus. Neuroscience 69:1031–1055. - PubMed
1. Bisogno T, Howell F, Williams G, Minassi A, Cascio MG, Ligresti A, Matias I, Schiano-Moriello A, Paul P, Williams EJ, Gangadharan U, Hobbs C, Di Marzo V, Doherty P (2003). Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain. J Cell Biol 163:463–468. - PMC - PubMed

Localization of diacylglycerol lipase-alpha around postsynaptic spine suggests close proximity between production site of an endocannabinoid, 2-arachidonoyl-glycerol, and presynaptic cannabinoid CB1 receptor - PubMed (original) (raw)