Apolipoprotein receptors belong to an evolutionarily conserved surface receptor family that has intimate roles in the modulation of synaptic plasticity and is necessary for proper hippocampal-dependent memory formation. The known lipoprotein receptor ligand Reelin is important for normal synaptic plasticity, dendritic morphology, and cognitive function; however, the in vivo effect of enhanced Reelin signaling on cognitive function and synaptic plasticity in wild-type mice is unknown. The present studies test the hypothesis that in vivo enhancement of Reelin signaling can alter synaptic plasticity and ultimately influence processes of learning and memory. Purified recombinant Reelin was injected bilaterally into the ventricles of wild-type mice. We demonstrate that a single in vivo injection of Reelin increased activation of adaptor protein Disabled-1 and cAMP-response element binding protein after 15 min. These changes correlated with increased dendritic spine density, increased hippocampal CA1 long-term potentiation (LTP), and enhanced performance in associative and spatial learning and memory. The present study suggests that an acute elevation of in vivo Reelin can have long-term effects on synaptic function and cognitive ability in wild-type mice.

Reelin is a large extracellular matrix protein that plays a pivotal role in embryonic neuronal migration. Reelin is produced by GABAergic interneurons in the adult brain and physically associates with the postsynaptic density, dendritic spines, and axons throughout the hippocampus and cortex (Pesold et al. 1999). Reelin activates a number of neuronal signal transduction pathways in the adult central nervous system (CNS) that subsequently modulate synaptic function and plasticity. Interneurons expressing Reelin are widely distributed in the adult mammalian brain at a period long after the decrease in Cajal-Retzius cells (D'Arcangelo et al. 1997; Pesold et al. 1998, 1999; Rodriguez et al. 2000; Pappas et al. 2001; Kubo et al. 2002). Disruption of either Reelin expression or the two known receptors of Reelin, Apolipoprotein E Receptor 2 (ApoER2) and Very-Low-Density Lipoprotein Receptor (VLDLR), results in associative and spatial learning defects, impairment of hippocampal long-term potentiation (LTP), and alterations in dendritic spine morphology (Trommsdorff et al. 1999; Weeber et al. 2002a). Organotypic hippocampal cultures of mutant Reeler mice have decreased spine density, which is rescued with recombinant Reelin application in a lipoprotein-dependent manner (Niu et al. 2008). Conversely, transgenic mice that overexpress Reelin present with hypertrophy of dendritic spines in the hippocampus (Pujadas et al. 2010). Wild-type organotypic hippocampal cultures chronically treated with Reelin (>5 d) results in increases of dendritic spine density and increased AMPA receptor insertion. Acute Reelin application (<20 min) enhances LTP in acute hippocampal slices from wild-type mice, an effect dependent on the presence of both ApoER2 and VLDLR (Weeber et al. 2002a). Reelin-dependent enhancement of LTP is associated with increased Ca2+ currents in CA1 pyramidal neurons and increased N-methyl-D-aspartic acid receptor (NMDAR) phosphorylation (Beffert et al. 2005; Qiu et al. 2006b). Extended Reelin exposure (>20 min) increases α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)-mediated synaptic responses through Phosphoinositide-3-kinase (PI3K)-dependent increases in AMPAR insertion, which is associated with a significant reduction of silent synapses (Qiu et al. 2006b).

Taken together, these findings support a role that Reelin signaling is important in overall synaptic function and can influence neuronal activity and cellular mechanisms underlying memory formation. The interpretation of in vitro experimentation makes it difficult to determine the long-term consequences of enhanced Reelin signaling in vivo on complicated processes such as cognitive ability. The present studies focus on acute in vivo activation of Reelin signaling and determine the long-lasting changes to synaptic morphology, physiology, and ultimately, processes of learning and memory.

Results

To determine if Reelin could be transported from the ventricle into the hippocampus, a single bilateral injection of Reelin or saline was given to 4-mo-old adult mice. We previously showed that perfusion of 5 nM Reelin onto acute hippocampal slices increased LTP (Weeber et al. 2002a). Thus, the injection volume of Reelin was determined to produce an average total hemisphere concentration of 5 nM. Immunohistochemical staining for Reelin revealed rapid uptake by the hippocampus 15 min following Reelin injection. Reelin levels remained elevated 3 h following Reelin injection throughout the entire hippocampus (Fig. 1). Although robust initially, Reelin levels were comparable to saline controls 5 d post-injection, suggesting that exogenous Reelin is maintained transiently.

View larger version: Download as PowerPoint Slide Figure 1. Reelin injection increased expression throughout the entire hippocampus. Reelin immunoreactivity was detected using the G10 anti-Reelin antibody (left column). Immunohistochemical analysis of Reelin revealed increased Reelin immunoreactivity in the hippocampus 15 min following injection, whereas saline injection resulted in no detectable differences. Reelin levels were found to be uniformly increased 3 h following injection and returned to baseline levels 5 d post-injection (scale bar: 50 µm).

To determine whether in vivo application of Reelin can result in specific Reelin signaling, we examined the activation state of the obligate downstream adaptor protein Disabled-1 (Dab1) at 15 min, 3 h, and 5 d following injection. Reelin induces tyrosine phosphorylation at Y220 by Src family tyrosine kinases (SFKs) in vitro (Keshvara et al. 2001; Ballif et al. 2003). Reelin injected mice showed pronounced increases in Dab1 phosphorylation at Tyrosine-220 (Y220) throughout the hippocampus at 15 min and 3 h (Fig. 2). Importantly, increases in Dab1 phosphorylation were less robust at 3 h than 15 min, which is consistent with rapid degradation of tyrosine phosphorylated Dab1 following Reelin stimulation (Arnaud et al. 2003).

View larger version: Download as PowerPoint Slide Figure 2. Reelin injection enhanced Dab1 phosphorylation throughout the entire hippocampus. Activation of Dab1 was determined with p-Tyr220 specific antibody. Immunohistochemical analysis of Dab1 activation revealed that Reelin injection resulted in increased Tyr220 phosphorylation of Dab1 at the 15-min time point, remained elevated through 3 h and returned to baseline after 5 d post-injection (scale bar: 50 µm).

Reelin enhances Ca2+ entry through NMDA receptors and results in increased Ser 133 phosphorylation and nuclear translocation of CREB (Beffert et al. 2006). Once in the nucleus, CREB promotes transcription of genes important for the formation of new synaptic connections and long-term memories (Huang et al. 1996; Martin et al. 1997; Pang and Lu 2004; Zhao et al. 2005). We detected active CREB pSer-33 was elevated in CA1, CA3, and dentate gyrus at 15 min and 3 h post-injection (Fig. 3). Similar to Dab1, phosphorylation levels of CREB were noticeably lower at 3 h and returned to baseline levels at 5 d post-injection. Increased CREB phosphorylation was also seen with quantitative Western blot analysis (Supplemental Fig. 1). To determine ApoER2 dependence of Reelin induced increases in Dab1 and CREB phosphorylation states, we injected ApoER2 KO with Reelin. No differences were detected to Dab1 or CREB phosphorylation compared to saline injected ApoER2 KO animals (data not shown). These findings confirm that in vivo application of purified recombinant Reelin can activate downstream signaling pathways similar to that seen with in vitro Reelin application.

View larger version: Download as PowerPoint Slide Figure 3. Reelin injection enhanced CREB phosphorylation throughout the entire hippocampus. Activation of CREB was determined with p-Ser133 specific antibody. Immunohistochemical analysis of CREB activation revealed that Reelin injection resulted in increased Ser133 phosphorylation of CREB at the 15-min time point, remained elevated through 3 h, and returned to baseline after 5 d post-injection (scale bar: 50 µm).

Considering chronic application of Reelin to hippocampal cultures increases spine density, we determined whether Reelin injection influenced dendritic spine formation. Spine density on the apical oblique (AO) and basal shaft (BS) dendrites of CA1 pyramidal neurons was quantified at 3 h and 5 d post-injection. Spine density was significantly increased in 5 d Reelin-injected mice compared to 3 h (Fig. 4). To determine if the presence of ApoER2 is required for Reelin-induced increases in spine density, ApoER2 KO mice were injected with Reelin bilaterally into the ventricles 5 d post-injection when the Reelin-dependent effect on spine density was observed. All Reelin-injected experimental groups were normalized to their respective saline control groups. At 5 d post-injection, we found that both AO and BS spine densities were significantly increased in Reelin-injected wild-type mice compared to both saline-injected wild-type and Reelin-injected ApoER2 KO mice (Fig. 5). The modest increase of spine density in Reelin-injected ApoER2 KO mice may be attributed to Reelin–VLDLR interactions and signaling. Taken together, these findings are consistent with Reelin application in cultured hippocampal slices and correlate increased spine density with the activation of Dab1 and CREB.

View larger version: Download as PowerPoint Slide Figure 4. Reelin increased spine density in pyramidal cells in area CA1. Mouse brains were Golgi stained and CA1 were imaged (n = 4 mice/group). (A) Representative AO dendrites at 3 h and at 5 d per group. (B) Averaged AO spine densities for each group at 3 h (saline: 15.21 ± 0.79, n = 25; Reelin: 15.51 ± 0.79, n = 28) and at 5 d (saline: 13.5 ± 0.60, n = 25; Reelin: 19.2 ± 0.86, n = 34). Reelin significantly increased AO spine density in area CA1 at 5 d but not 3 h post-injection. (C) Representative BS dendrites at 3 h and at 5 d for each group. (D) Averaged BS spine densities for each group at 3 h (saline: 15.75 ± 0.76, n = 25; Reelin: 15.30 ± 0.71, n = 33) and at 5 d (saline: 13.64 ± 0.85, n = 28; Reelin: 17.64 ± 0.70, n = 28). Reelin significantly increased BS spine density in area CA1 at 5 d but not 3 h post-injection. (Data expressed as mean ± SEM; *P < 0.0005.)

View larger version: Download as PowerPoint Slide Figure 5. Reelin significantly increased spine density in wild-type mice compared to ApoER2 KO mice 5 d post-injection in area CA1. Spine increases are expressed as a percentage of experimental saline controls. (A) Representative AO dendrites of both Reelin-treated groups at 5 d post-injection. (B) Averaged AO percent dendritic spine increase for each group (wild-type saline: 100.0 ± 4.4%; wild-type Reelin: 142.3 ± 6.5%; ApoER2 KO saline: 100 ± 4.3%; ER2 KO Reelin: 113.6 ± 3.7%). Reelin significantly increased AO spine density in area CA1 compared to saline- or Reelin-injected ER2 − /− mice. (C) Representative BS dendrites of both Reelin-treated groups at 5 d post-injection. (D) Averaged BS percent dendritic spine increase for each group (wild-type saline: 100.0 ± 6.2%; wild-type Reelin: 130.0 ± 5.1%; ApoER2 KO saline: 100 ± 6.5%; ER2 KO Reelin: 110.8 ± 7.6%). Reelin significantly increased BS spine density in area CA1 compared to saline- or Reelin-injected ER2 − /− mice (*P < 0.05; **P < 0.01; ***P < 0.001).

Our previous report of increased hippocampal LTP following acute application of Reelin is likely a result of Reelin-dependent changes in NMDAR regulation, as well as localized signal transduction activation (Qiu et al. 2006b). However, 5 d following Reelin injection, post-translational modifications of ligand-gated ion channels and Reelin-activated signaling systems would be expected to return to homeostatic levels. It is possible that the observed altered spine density following the single injection of Reelin is sufficient to change overall synaptic function in the hippocampus. Thus, we determined if synaptic transmission or plasticity in area CA1 is altered 5 d following Reelin injection. Reelin-injected mice showed significantly enhanced theta-burst stimulation (TBS)-induced LTP (Fig. 6A–B). Examination of basal synaptic transmission revealed no significant differences of field excitatory post-synaptic potential (fEPSP) slopes between groups in response to equivalent amounts of input (Fig. 6C) or alteration in fiber volley amplitude (data not shown). Also, no changes in presynaptic function were supported by quantifying short-term plasticity evaluated by paired-pulse facilitation (PPF). No significant changes were observed in PPF testing between experimental groups (Fig. 6D), further suggesting that changes in LTP are primarily due to postsynaptic modification following TBS.

View larger version: Download as PowerPoint Slide Figure 6. Reelin enhances hippocampal synaptic plasticity. Mice were sacrificed 5 d following single, bilateral injections for electrophysiology experiments. LTP was induced with TBS (five bursts of 200 Hz separated by 200 msec, repeated six times with 10 sec between the six trains; arrow) after 20 min of baseline recording and changes in fEPSP slope are expressed as a percentage of baseline. (A) Representative fEPSP traces from both saline (white) and Reelin (black) injected hippocampi. (B) Reelin injection enhanced LTP in area CA1. The last 5 min of fEPSPs slope recordings were averaged (bar) for both saline (n = 9) and Reelin (n = 10) injected (*P < 0.05). (C) Output field analysis following increasing field stimulation fit with nonlinear regression. There were no significant differences between experimental groups. (D) PPF was induced with the use of paired pulses given with an initial delay of 20 msec and the time to the second pulse was increased 20 msec incrementally until a final delay of 300 msec was reached. There was no significant difference between experimental groups.

Initially, mice were tested in the open field and elevated plus maze (EPM) to determine if Reelin injection alters somatosensory input and anxiety levels. We found that Reelin injection had no effect on the mice to explore either the open field or EPM (Fig. 7A,B). These results indicate that Reelin injection has no effect on anxiety levels in these mice. Furthermore, any significant differences found in spatial or associative learning through Reelin injection would not be attributable to alterations in anxiety, but rather cognitive function.

View larger version: Download as PowerPoint Slide Figure 7. There is no effect of Reelin on general locomotor or anxiety levels in wild-type mice. (A) After 5 d post-injection of saline (n = 7) or Reelin (n = 6), mice underwent open-field testing as a locomotor and general anxiety control for behavioral testing. Data represent the ratio of time spent (sec) in the open field vs. the perimeter of the field. There were no significant differences between experimental groups. (B) After 5 d post-injection, mice underwent elevated plus maze testing as a general anxiety control. Data represent the ratio of time spent (sec) in the open areas vs. the closed arms of the elevated maze. There were no significant differences between experimental groups.

To determine if Reelin supplementation enhances spatial learning and memory, mice were tested in the hidden platform water maze (HPWM) task. Mice were trained using a spaced experimental paradigm of four trials per day with an intertrial interval of 1 h. Reelin-injected mice required less time to find the hidden platform on training days 1, 3, and 4, showing that Reelin injection improved spatial learning in the HPWM (Fig. 8A). Of particular interest was the significant difference exhibited by Reelin-injected animals on day 1. Closer examination of each trial on day 1 revealed Reelin-injected mice had significantly shorter latencies on trials 3 and 4 compared to the first trial (Fig. 8B). Furthermore, by trials 3 and 4 of day 1, Reelin-treated mice found the platform in the same amount of time as the saline-treated mice during cued testing (data not shown).

View larger version: Download as PowerPoint Slide Figure 8. Reelin supplementation enhanced spatial learning and memory. Mice began training in the HPWM 5 d post-injection of saline (white, n = 7) or Reelin (black, n = 5). Mice were trained in the HPWM for 4 d, four trials per day. On day 5, the platform was removed and a 60-sec probe trial was conducted. On trial days 6 and 7, the platform was moved to the opposite quadrant. (A) Reelin significantly reduced latency to the platform during HPWM training. (B) Analysis of day 1 training reveals Reelin reduced latencies to the platform after the second trial compared to the initial trial. Reelin significantly lowered latencies on trials 3 and 4 compared to saline-injected animals. (C) Reelin increased target quadrant entries during the probe trail compared to saline-treated animals (*P < 0.05; #P < 0.01).

Of particular interest was the effect of Reelin injection on memory retention in the HPWM. Reelin-injected mice had a significantly greater amount of target quadrant entries compared to saline-injected mice during the probe trial conducted on day 5, indicating enhanced memory retention of platform location (Fig. 8C). Reelin-injected mice retrained to find a different platform location (opposite quadrant) on day 6 continued to show a significant decrease in latency to the platform on the second day of training (Fig. 8A). Taken together, Reelin-injected mice learned more quickly, retained memory more efficiently, and relearned more quickly.

We next examined hippocampal-dependent associative fear conditioned learning and memory. Animals were trained with a standard two-shock protocol as previously described (Weeber et al. 2002a). Three separate groups of mice were tested for freezing to the context at either 1, 24, or 72 h after training. Reelin- injected mice exhibited enhanced context-dependent freezing at both 24-h and 72-h time points, but not at the 1-h time point (Fig. 9). Taken together, these data demonstrate that Reelin supplementation in wild-type mice enhances both associative and spatial learning and memory with a single injection of Reelin 5 d prior to the start of training.