Article Figures & Data
Figures
- Figure 1.
NAPE-PLD mRNA is expressed in selected brain regions at moderate-to-high levels. A , In situ hybridization using a digoxigenin-labeled riboprobe against the mouse NAPE-PLD mRNA sequence highlights that granule cells in the dentate gyrus may have the highest expression of NAPE-PLD in the forebrain. Note that a relatively high expression level is also found in the CA3 pyramidal neurons, whereas modest labeling is visible in selected areas of the neocortex, the piriform cortex, the thalamus, and the hypothalamus. B , In contrast, no labeling was found on sections derived from knock-out animals. Sections from wild-type and knock-out animals were mixed in the incubation wells and were processed together throughout the reactions. C–E , High-magnification light micrographs show pyramidal neurons in layer II of the piriform cortex ( C ), neurons in higher-order thalamic nuclei ( D ), and neurons in the ventromedial hypothalamic nuclei (VMH; E ). LD, Laterodorsal thalamic nucleus; LP, lateral posterior thalamic nucleus; Po, posterior thalamic nuclei; MD, mediodorsal thalamic nucleus; VPM, ventral posteromedial thalamic nucleus. Scale bars: A , B , 1 mm; C , E , 100 μm; D , 200 μm.
- Figure 2.
A , In situ hybridization reveals high levels of NAPE-PLD mRNA in granule cells and CA3 pyramidal neurons in wild-type animals. C , Immunocytochemistry for NAPE-PLD results in the strongest labeling in the hilus and in stratum lucidum, where mossy fibers arborize. B , D , The specificity of both reactions is confirmed in knock-out animals. Scale bars, 200 μm.
- Figure 3.
A , Immunocytochemistry for NAPE-PLD visualizes the laminar structure of the hippocampus, which is determined by the topography of excitatory pathways. The strongest labeling is found in the hilus (h) of the dentate gyrus. Dense NAPE-PLD immunoreactivity is present also in stratum moleculare (s.m.) (more profoundly in its inner third) and in the strata oriens (s.o.) and radiatum (s.r.) of the CA1 subfield, whereas in stratum lacunosum-moleculare (s-l.m.), immunostaining for NAPE-PLD did not reach detection threshold. B , D , At higher magnification, NAPE-PLD immunostaining reveals a dense punctate pattern in the CA1 stratum radiatum ( B ) and a very dense punctate labeling in the hilus of the dentate gyrus ( D ). C , E , Immunogold particles (arrows) representing the precise subcellular localization of NAPE-PLD are found presynaptically in excitatory axon terminals both in the stratum radiatum (in Schaffer collaterals; b in C ) and in the hilus (mossy terminals; b in E ), whereas postsynaptic structures [dendritic spines (sp) in C and thorny excrescences (te) in E ] are immunonegative. DG, Dentate gyrus; s.p., stratum pyramidale; s.g., stratum granulosum. Scale bars: A , D , 50 μm; B , 10 μm; C , E , 200 nm.
- Figure 4.
A 1, A 2, High magnification of serial sections from a mossy fiber terminal demonstrates that NAPE-PLD is present on cisternae of SER, but rarely on synaptic vesicles or on the plasma membrane. B , A similar subcellular localization is found in Schaffer collaterals in CA1. C , Quantitative analysis of subcellular immunogold distribution confirms the preferential localization of NAPE-PLD on cisternae of SER. Note that size limitations in immunogold labeling and the dense packing of synaptic vesicles (V) in mossy terminals exclude unequivocal determination of gold particle position in several cases; this category is indicated as SER/V. Nevertheless, the ratio of gold particles found on either SER or V is significantly different (Mann–Whitney U test; n = 4 mice indicated as m1–m4). PM, Plasma membrane. Scale bar, 100 nm.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Legend
- supplemental material - Supplemental Figure
