Fast-acting antidepressant-like effects of Reelin evaluated in the repeated-corticosterone chronic stress paradigm

Abstract

The present report examines the effects of repeated or single intrahippocampal Reelin infusions on measures of depressive-like behavior, cognition, and hippocampal neurogenesis in the repeated-corticosterone (CORT) paradigm. Rats received subcutaneous injections of CORT for 3 weeks and Reelin was infused through an inserted canula in the left hippocampus on days 7, 14, and 21, or only on day 21 of CORT injections. CORT increased immobility in the forced-swim test and impaired object-location memory. Notably, these effects were reversed by both repeated and single-Reelin infusions. CORT decreased both the number and complexity of doublecortin-labeled maturing newborn neurons in the dentate gyrus subgranular zone, and a single-Reelin infusion increased the number but not complexity of newborn neurons, while repeated Reelin infusions restored both. Injection of the AMPA antagonist CNQX blocked the rescue of the behavioral phenotype by Reelin but did completely block the effects of Reelin on hippocampal neurogenesis. Reelin is able to rescue the deficits in AMPA, NMDA, GABA_A receptors, mTOR and p-mTOR induced by CORT. These novel results demonstrate that a single intrahippocampal Reelin infusion into the dorsal hippocampus has fast-acting antidepressant-like effects, and that some of these effects may be at least partially independent of Reelin actions on hippocampal neurogenesis.

Fig. 1. Experimental design, and evaluation of behavioral tests.

a Schematic representation of the experimental design used for the study. Stereotaxic surgery was performed and a chronic indwelling cannula was inserted into the left dorsal hippocampus. Animals were weight-matched and received 21 days of 40mg/kg of corticosterone (CORT) or vehicle injections. A subset of animals received infusions of 1 µg/µl of Reelin either on days 7, 14, and 21 of CORT/vehicle injections, or only on day 21. All rats underwent the forced-swim test (FST) on day 21, and a subset of rats were immediately sacrificed (time 1). The remaining rats then underwent the object-location memory paradigm (OBL) from days 23 to 28, and were then sacrificed (time 2). All experimental groups were evenly distributed throughout both times 1 and 2. Shown in panel C, a new group of animals underwent stereotaxic surgery and an indwelling cannula was implanted into left dorsal hippocampus. Animals were weight-matched and received 21 days of 40 mg/kg of CORT or vehicle injections. A subset of animals received infusions of 1 µg/µl Reelin on day 21, and another subset of animals received an infusion of 1 µg/µl of CNQX 30 min prior to the FST. b Mean body weight through days 1–21, collapsed across both times 1 and 2. Panel A shows that CORT-treated and CORT repeated-Reelin rats weighed less than vehicle, vehicle Reelin single, and vehicle repeated-Reelin rats on day 14 (p < 0.05) and 21 (p < 0.05) and significantly less than vehicle and vehicle Reelin single rats on day 7 (p < 0.05), and CORT Reelin single rats weighed significantly less than vehicle and vehicle Reelin single rats on days 7 and 14 (p < 0.05), and significantly less than vehicle, vehicle Reelin single, and vehicle repeated-Reelin rats on day 21 (p < 0.05). B–D: CORT had significant effects on the FST and OBL, and treatment with Reelin reversed this in a fast-acting manner. Data are collapsed across times 1 and 2. Panel B shows the effects of treatment on time spent immobile in the FST. CORT-treated rats spent significantly more time immobile than all other groups (p < 0.05). Panel C shows the effects of treatment on time spent struggling. CORT-treated rats spent significantly less time struggling than all other groups (p < 0.05). Panel D shows the effects of treatment on time spent swimming. CORT-treated rats spent significantly less time swimming than all other groups (p < 0.05). CORT had significant effects on OBL memory, and Reelin restored this. Panel E shows the discrimination ratio for the OBL variant. CORT-treated rats had a significantly lower discrimination ratio than all other groups (p < 0.05). All data are represented as means ± standard error of the mean.

Fig. 2. Effects of CORT and Reelin on DCX-ir cells.

Effects of Reelin on hippocampal neurogenesis at times 1 (a–c) and 2 (d). CORT had significant effects on hippocampal neurogenesis. a Representative photomicrographs of doublecortin expression in the granule cell/subgranular zone. b Representative images of DCX-ir cell maturation that have been used in our categorization studies. c, d Effects of treatment on the number of doublecortin-ir cells in the granule cell/subgranular zone at times 1 and 2, respectively (see Fig. 1). CORT-treated rats had significantly fewer doublecortin-ir cells in the ipsilateral hemisphere than vehicle Reelin single, CORT Reelin single, vehicle repeated-Reelin, and CORT repeated-Reelin animals. In the contralateral hemisphere, CORT-treated rats had significantly fewer doublecortin-ir cells than vehicle, vehicle Reelin single, vehicle repeated-Reelin, and CORT repeated-Reelin animals (these effects are more evident, i.e. stronger, at time 2). Similarly, the reversal of CORT-induced alterations in the quantified categorization of dendritic complexity induced by Reelin infusions is evident already at time 1 and stronger at time 2. All data are represented as means ± standard error of the mean. VRS vehicle Reelin single, CRS CORT Reelin single, VRR vehicle repeated-Reelin, CRR CORT repeated-Reelin.

Fig. 3. CORT had significant effects on depressive-like behavior, treatment with Reelin restored this, and CNQX abolished Reelin’s antidepressant effect.

CORT and CORT Reelin CNQX-treated rats spent significantly more time immobile than all other groups (a) (p values <0.05). b Effect of treatment on time spent struggling. c Effect of treatment on time spent swimming. CORT-treated rats spent significantly less time swimming than vehicle, vehicle Reelin single, and CORT Reelin single-treated rats (p values <0.05), and CORT Reelin CNQX-treated rats spent significantly less time swimming than vehicle, vehicle Reelin single, and CORT Reelin single-treated rats (p values <0.05). All data are represented as means ± standard error of the mean.

Fig. 4. Effects of intrahippocampal Reelin and CNQX-treatment on hippocampal neurogenesis.

CORT had significant effects on hippocampal neurogenesis. a Representative photomicrographs of doublecortin expression in the granule cell/subgranular zone (scale bar = 200 μm). A higher magnification image is shown in the insets (scale bar = 50 μm). b Effects of treatment on the number of doublecortin-ir cells in the granule cell/subgranular zone. Overall, CORT-treated rats had significantly fewer doublecortin-ir cells than CORT Reelin single and CORT Reelin single CNQX-treated rats in the ipsilateral hemisphere (p values <0.05). c Quantified categorization of dendritic complexity using doublecortin staining. All data are represented as means ± standard error of the mean. VRS vehicle Reelin single, CRS CORT Reelin single, VRCNQX vehicle Reelin single CNQX, CRCNQX CORT Reelin single CNQX.

Fig. 5. Western blot evaluation of PSD-95 and mTOR expression upon incubation with Reelin of hippocampal synaptosomes from CORT-treated animals.

Optical density analysis demonstrates that CORT decreases expression of mTOR, p-mTOR, active mTOR ratio (p-mTOR/mTOR), and PSD-95, and that Reelin incubations are able to rescue these deficits in a concentration-dependent manner.