Developmental regulation of synaptotagmin I, II, III, and IV mRNAs in the rat CNS

Abstract

Synaptotagmin I is an abundant synaptic vesicle protein that has an essential function in mediating Ca2+-triggered neurotransmitter release. We have analyzed the distribution of four neural synaptotagmin isoforms during postnatal development of the rat CNS by in situ hybridization. Synaptotagmin I, II, III, and IV genes have distinct patterns of spatiotemporal expression except in cerebellum granule cells, where the four transcripts were detected during the formation of parallel fiber/Purkinje cell synapses. Throughout development synaptotagmin I mRNAs were widely expressed in brain, whereas synaptotagmin II transcripts were predominant in spinal cord. At all stages synaptotagmin III mRNAs were expressed uniformly in most neurons examined, although at a low level. Synaptotagmin I, II, and III gene expressions mainly increased during development and persisted in adulthood, mirroring neuronal differentiation. Conversely, synaptotagmin IV transcripts were predominant during perinatal development in a heterogeneous population of neurons and subsequently were expressed uniformly at a low level. Intense labeling was observed in the hippocampal CA3 field and in the subiculum, but not in the CA1 field, of the newborn rat. In cerebral cortex, lamina-specific labeling was detected with a high expression in cell layer V. Only a small number of Purkinje cell clusters were labeled in the flocculus and paraflocculus of the cerebellum. Heterogeneous sets of neurons expressing synaptotagmin IV gene also were observed in spinal cord. We thus speculate that synaptotagmin IV may a play a role in the development of the mammalian nervous system.

Fig. 1.

Overview of postnatal expression of synaptotagmin I, II, III, and IV transcripts in rat brain. In situhybridization of synaptotagmin I (A–E), II (F–J), III (K–O), and IV (P–T) mRNAs in horizontal sections of newborn (P0), 6- (P6), 15- (P15), and 20-d-old (P20) and adult (Ad) rat brain. Exposure was on Kodak Biomax film for 2 weeks with synaptotagmin I and IV probes, 5 weeks with synaptotagmin II, and 6 weeks with synaptotagmin III probes. Negative digitalized images were produced by scanning autoradiographic films. Synaptotagmin I, II, and IV mRNA distributions were obtained in the same experiment. Cb, Cerebellum; CPu, caudate putamen; Cx, cortex; DG, dentate gyrus; Gl, glomerular layer; Hi, hippocampus; IC, inferior colliculi; Igr, internal granule cell layer; M, mitral cell layer;Mhb, medial habenula; OB, olfactory bulb;Rt, reticular thalamic nucleus; S, septum; SC, superior colliculi; Th, thalamus. Scale bar, A–T, 5 mm.

Fig. 2.

Overview of postnatal expression of synaptotagmin I, II, III, and IV transcripts in rat spinal cord. In situ hybridization of synaptotagmin I (A–C), II (D–F), III (G–I), and IV (J–L) mRNAs on newborn (P0), 6- (P6), and 20-d-old (P20) sections. Exposures were on Kodak Biomax film for 7 weeks. The dorsal horns are oriented toward the top of each photomicrograph. Scale bar, A–L, 1 mm.

Fig. 3.

Differential distributions of synaptotagmin I, II, and IV transcripts in the developing olfactory bulb. High-power bright-field photomicrographs of 6- (P6), 15- (P15), and 20-d-old (P20) rat brain sections show the distribution of synaptotagmin I (A–C), II (D–F), and IV (G–I) mRNAs. Exposure times of the emulsion-coated slides were 6 weeks. Gl, Glomerular layer; Igr, internal granule cell layer.Arrowheads point to the mitral cell layer (M). Scale bar, A–I, 35 μm.

Fig. 5.

Distinct distributions of synaptotagmin I, II, and IV transcripts in the developing rat hippocampus. High-power bright-field photomicrographs through the CA3 cell layer illustrate the distribution of synaptotagmin I (A–C), II (D–F), and IV(G–I) on 4- (P4), 13- (P13), and 20-d-old (P20) rat brain sections. Exposure times were 6 weeks.Arrowheads highlight labeled CA3 cells. Scale bar,A–I, 23 μm.

Fig. 7.

Differential distributions of synaptotagmin I, II, and IV transcripts in the developing cerebellum. High-power bright-field photomicrographs display the distribution of synaptotagmin I (A–C), II (D–F), and IV (G–I) transcripts on 6- (P6), 15- (P15), and 20-d-old (P20) rat brain sections. Exposure times were 6 weeks.EGL, External germinal layer; mol, molecular layer; Pj, Purkinje cell layer;Gr, granule cell layer. Arrowheads point to some highly labeled cells in the granule cell layer.Arrows show the labeling of the premigratory zone of the external plexiform layer. Scale bars: A, D, G, 14 μm;B, C, E, F, H, I, 23 μm.

Fig. 4.

Distinct expression of synaptotagmin I and IV transcripts at P4 in cortex (A,D), hippocampus (B, E), and cerebellum (C, F). Bright-field photomicrographs of coronal sections display the distribution of synaptotagmin I (A–C) and synaptotagmin IV (D–F) transcripts. Exposure times were 6 weeks. A, D, Cortex; B,E, hippocampus; C, F, cerebellum. An arrowhead points to some highly labeled cells in F. CA1, CA1 cell layer;CA3, CA3 cell layer; DG, dentate gyrus;EGL, external germinal layer (arrowheadsin C and F); Fl, flocculus. Ventral side of the cerebellum is oriented toward theleft of photomicrographs C andF. Scale bars: A, D, 135 μm; B, C, E, F, 240 μm.

Fig. 6.

Cellular expression of synaptotagmin IV transcripts at P4 in the flocculus of the cerebellum. Shown are bright-field photomicrographs of sagittal sections hybridized with the synaptotagmin IV antisense (A) and sense (B) probes. Exposure times were 6 weeks.EGL, External germinal layer; Pj, Purkinje cell layer. Scale bar, A, B, 17 μm.