Reelin regulates postnatal neurogenesis and enhances spine hypertrophy and long-term potentiation

Abstract

Reelin, an extracellular protein essential for neural migration and lamination, is also expressed in the adult brain. To unravel the function of this protein in the adult forebrain, we generated transgenic mice that overexpress Reelin under the control of the CaMKIIalpha promoter. Overexpression of Reelin increased adult neurogenesis and impaired the migration and positioning of adult-generated neurons. In the hippocampus, the overexpression of Reelin resulted in an increase in synaptic contacts and hypertrophy of dendritic spines. Induction of long-term potentiation (LTP) in alert-behaving mice showed that Reelin overexpression evokes a dramatic increase in LTP responses. Hippocampal field EPSP during a classical conditioning paradigm was also increased in these mice. Our results indicate that Reelin levels in the adult brain regulate neurogenesis and migration, as well as the structural and functional properties of synapses. These observations suggest that Reelin controls developmental processes that remain active in the adult brain.

Figure 1.

Generation and characterization of conditional Tg1/Tg2 transgenic mice. a, Transgenic mice that overexpress Reelin were based on the Tet-off regulated binary system: the Tg1 transgene contains the tTA transactivator set under the control of pCaMKIIα, while the Tg2 transgene contains rlM controlled by the tetO promoter. Double transgenic mice (Tg1/Tg2) express Reelin in neurons expressing CaMKIIα; transgene expression can be switched off by doxycycline administration, which inactivates tTA transactivator. b, Western blot experiments showing Reelin, Dab1, and actin protein levels in protein lysates obtained from control and Tg1/Tg2 adult hippocampus (HP), cerebral cortex (CX), and striatum (STR). Reelin levels are increased in Tg1/Tg2 (T) mice compared with control littermates (C) or reeler animals (R). Dab1 levels are slightly reduced in Tg1/Tg2 mice. c, Immunohistochemical detection of Reelin shows that expression of this protein in control adult mice is restricted to a subset of interneurons distributed throughout the cortex and hippocampal layers, while the striatum shows a diffuse staining (top). In Tg1/Tg2 mice, overexpression of Reelin is observed in hippocampal pyramidal cells and in granule cells of the dentate gyrus (arrows in bottom left panel); Reelin is also expressed in neocortical pyramidal cells (bottom middle) and in striatal neurons (bottom right). I–VI, Cortical layers; CA1–CA3, hippocampal regions; CPu, caudate–putamen nucleus; DOX, doxycycline; H, hilus; LV, lateral ventricle; ML, molecular layer; SP, stratum pyramidale; WM, white matter. Scale bars: c (left): 200 μm; c (middle and right): 100 μm.

Figure 2.

Double-immunohistofluorescence staining in tissue sections from adult Tg1/Tg2 mice. a–c, Double immunodetection of Reelin (green) and the neuronal marker NeuN (red) in hippocampal tissue sections from Tg1/Tg2 mice counterstained with DAPI (blue). a, Low-power micrographs showing Reelin-expressing cells distributed throughout the hippocampal layers, including the CA1 pyramidal neurons and the granule cells. High-power micrographs from CA1 (b) and DG (c) regions, demonstrating that cells expressing Reelin are also positive for NeuN. d, Double immunodetection of Reelin (green) and the glial marker GFAP (red) in hippocampal tissue sections from Tg1/Tg2 mice counterstained with DAPI. The staining patterns for Reelin are not coincident with those for GFAP. Insets, High-power micrographs demonstrating that GFAP and Reelin immunoreactivities do not overlap. e, f, Double immunodetection of Reelin (green) and neuronal marker NeuN (red) in tissue sections from the striatum (STR) (e) and neocortex (CX) (f) of Tg1/Tg2 mice counterstained with DAPI (blue). In both regions, Reelin-expressing cells are also NeuN positive. II–III, Cortical layers; CA1–CA3, hippocampal regions; CPu, caudate–putamen nucleus; H, hilus; ML, molecular layer; SP, stratum pyramidale. Scale bars: a, d, 200 μm; b, c, e, f, 10 μm.

Figure 3.

Impairment of neural migration, but not neurogenesis, in the SVZ and RMS. a, Representative images from the adult SVZ immunostained against BrdU after BrdU pulses 1 d earlier. Control and Tg1/Tg2 sections were counterstained with Nissl dye. b, Quantification of BrdU-positive cells in the SVZ/RMS region revealed no differences in the number of BrdU-positive cells in control and Tg1/Tg2 mice, either in the SVZ or in the RMS. c, Representative images from OB sections immunostained against BrdU, from control and Tg1/Tg2 mice BrdU injected 20 d before being killed. Note the reduced numbers of stained cells in the GCL. d, Densities of BrdU-labeled neurons in the GCL and PGC layers of the OB, demonstrating decreased numbers of BrdU-positive cells in the GCL of Tg1/Tg2 mice. e, Distribution of TH-immunoreactive cells in control and in Tg1/Tg2 OB tissue sections from 12-month-old mice. In controls, TH-positive cells are restricted to the PGC layer; in Tg1/Tg2 sections ectopic TH-positive neurons are detected in the GCL (arrows in right panel). The inset depicts ectopic TH-positive neurons. f, Density of ectopic TH-immunoreactive neurons in the GCL of control and Tg1/Tg2 mice at different ages; notice the marked increment at older ages. CC, Corpus callosum; DiV, days in vivo; PGC, periglomerular cell layer; LV, lateral ventricle; EPL, external plexiform layer; MCL, mitral cell layer; STR, striatum. Data are presented as mean ± SEM; ***p < 0.001; Student's t test. Scale bars: a, c, e, 100 μm.

Figure 4.

Neurogenesis and migration in the hippocampal SGZ. a, Sections from P25 aged mice immunostained with BrdU antibodies. Mice were injected with BrdU at P15. Control mice show most BrdU-positive cells near the SGZ layer; Tg1/Tg2 mice show a wider distribution of BrdU-positive cells in the GL. b, Densities of BrdU-positive cells in P15-old mice injected with BrdU pulses and killed after 24 h, 10 d, or 20 d. Note increased numbers of BrdU-labeled cells in Tg1/Tg2 mice. c, Low-power micrographs from control and Tg1/Tg2 hippocampal sections illustrating higher numbers of DCX-positive cells in the dentate gyrus of Tg1/Tg2 mice. d, Counts of PSA-NCAM- and DCX-positive neurons in the dentate gyrus of 5-month and 12-month-aged mice. e, g, Photomicrographs demonstrating the radial distribution of BrdU-positive cells and DCX-immunoreactive cells in control and Tg1/Tg2 mice; note the wider distribution of immunopositive neurons in Reelin-overexpressing mice. f, h, Histograms showing radial distribution of BrdU- and DCX-immunopositive neurons in the GL of control and Tg1/Tg2 mice. Notice that in Tg1/Tg2 mice, many neurons are not restricted to the inner GL. DiV, Days in vivo; H, hilus; ML, molecular layer; P15, postnatal day 15. Data are presented as mean ± SEM; *p < 0.05; ***p < 0.001; Student's t test. Scale bars: a, c, 100 μm; e, g, 50 μm.

Figure 5.

BrdU-labeled cells are NeuN-positive neurons. a, b, Confocal photomicrographs showing BrdU/NeuN double-labeled neurons (arrows) in the dentate gyrus of P35-old wt and Tg1/Tg2 mice injected with BrdU 20 d before being killed. High-magnification, single-section confocal images are illustrated (b). c, Histogram showing that densities of BrdU/NeuN neurons increase in the dentate of Tg1/Tg2 mice. H, Hilus; ML, molecular layer. Data are presented as mean ± SEM; ***p < 0.001; Student's t test. Scale bar: a, 50 μm; b, 20 μm.

Figure 6.

Fine structural features of synaptic contacts in the hippocampus. a, b, Electron micrographs illustrating axon terminals and synaptic contacts (arrows) in the IML of control (a) and Tg1/Tg2 (b) mice. Note the higher complexity of synaptic boutons and dendritic spines in transgenic mice. c, d, Density of synaptic contacts (c) and percentage of synaptic terminals establishing at least two contacts (d) in different hippocampal layers in Tg1/Tg2 mice and littermate controls. e, Density of dendritic spines receiving at least one synaptic contact in different hippocampal layers. at, Axon terminal. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; Student's t test. Scale bars: a, b, 0.5 μm.

Figure 7.

Doxycycline (DOX) treatment (1 week) reverses the synaptic phenotype in Tg1/Tg2 mice. a, b, Electron micrographs illustrating axon terminals and synaptic contacts (arrows) in the IML of Tg1/Tg2 (a) and DOX-treated Tg1/Tg2 (b) mice. Note that the complexity of synaptic boutons and dendritic spines in transgenic mice is reversed upon DOX treatment. c, d, The density of synaptic contacts (c) and the percentage of synaptic terminals establishing at least two contacts (d) are reversed in Tg1/Tg2 mice treated with doxycycline. e, DOX treatment of Tg1/Tg2 mice leads to a marked reduction in the density of dendritic spines receiving at least one synaptic contact, reaching values lower than those observed in control mice. at, Axon terminal. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; Student's t test. Scale bars: a, b, 0.5 μm.

Figure 8.

Three-dimensional serial electron microscopic reconstructions of dendrites in the SR of CA1 region in control and Tg1/Tg2 mice, illustrating hypertrophy of dendritic spines in Tg1/Tg2 mice. a–c, e–g, Examples of serial electron micrographs in which the dendritic shaft and spines originating from it have been colored in green; numbers point to examples of reconstructed dendritic spines in d and h. d, h, Three-dimensional reconstructions of the identified dendritic segments shown in a–c and e–g; note the large hypertrophy of spines in h, compared to controls (d). Dendritic spine heads with sizes below and above 0.4 μm in diameter are represented by small and large arrows, respectively. Synaptic contacts are drawn in red. Dendritic spines in electron micrographs are labeled by numbers. Scale bars: a–c, e–g, 1 μm; d, h, 1 μm.

Figure 9.

In vivo induction of LTP in the CA1 area after electrical stimulation of the Schaffer collaterals in control and Tg1/Tg2 mice. a, Individual mice were subjected to paired pulses (40 ms interstimulus interval) presented at the CA3–CA1 synapse at increasing intensities of stimulation. PPF is observed in both control (*p ≤ 0.05) and Tg1/Tg2 (*p ≤ 0.05) mice at low stimulation intensities (≤0.2 mA). Control mice (left) switch from PPF to paired-pulse depression (PPD) at certain intensity values once they have reached a plateau of global synapse response [fEPSP¹⁺² represents the addition of the first (fEPSP¹) and the second (fEPSP²) evoked field synaptic responses]. Tg1/Tg2 mice (right) also reach a plateau at similar intensity values, but inversion from PPF to PPD does not occur (*p ≤ 0.05). b, fEPSP profiles evoked by paired pulses collected from representative animals at intermediate (0.16 mA; i.e., ∼30% of fEPSP¹⁺² asymptotic values) and high (0.24 mA) stimulus intensities. Note that controls (left) and Tg1/Tg2 mice (right) show similar profiles and equal PPF at the intermediate intensity level, but not at the high one. Calibrations are as indicated. c, fEPSP slopes evoked during the whole LTP experiment given as a percentage of the baseline (100%) values. Immediately after, fEPSPs were recorded for 2 h (day 1). To further check LTP evolution, fEPSPs were recorded for 15 min during the following days (2–8). Note that Tg1/Tg2 mice present a significantly (F_(79,136) = 5.83; p ≤ 0.05) larger LTP induction that is still present for the next 7 d. Data are presented as mean ± SEM. d, Representative fEPSP profiles from individual animals collected from control and T1/Tg2 mice during LTP evolution: baseline and 2 h (1), 24 h (2), and 48 h (3) after HFS. Illustrations are superimposed to show the time course evolution of representative individual profiles. e, Mean fEPSP slopes obtained during 15 min of recording for the two groups of animals (n = 15 each) were quantified at different times during LTP evolution. Data are presented as mean ± SEM (F_(8,72) = 12,452; *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 10.

Learning curves and evolution of CA3–CA1 extracellular synaptic field potentials for control and Tg1/Tg2 mice. a, A schematic representation of the classical conditioning paradigm, illustrating CS and US stimuli and the moment at which a single pulse (100 μs; square; biphasic) was presented to Schaffer collaterals (St. Hipp.). An example of an EMG recording from the orbicularis oculi (O.O.) muscle obtained from the 10th conditioning session is illustrated, as well as an extracellular recording of hippocampal activity from the same animal, session, and trial. b, Evolution of the increment (% over baseline values) of eyelid conditioned responses during the whole conditioning protocol. For both groups of animals, the increment in conditioned responses was significantly larger than values collected during habituation sessions at the indicated sessions (F_(16,144) = 14,810; *p < 0.001). Moreover, Tg1/Tg2 mice presented larger increases (p ≤ 0.05) of conditioned responses than controls from the fifth to the tenth conditioning sessions. c, Representative fEPSPs recorded in the CA1 area following a single pulse presented to the ipsilateral Schaffer collaterals 300 ms after CS presentation. fEPSPs were collected from both control and Tg1/Tg2 groups. fEPSP slopes were significantly larger than baseline values at the indicated sessions (F_(16,144) = 14,375; *p < 0.001). fEPSPs evoked in Tg1/Tg2 mice reached larger slopes than controls from the fifth conditioning session to the fifth extinction one. d, Superimposed fEPSP profiles corresponding to habituation session 1 (baseline) and from the 10th conditioning session (conditioned). Mean percentage values are followed by ±SEM.