Decreased proliferation of adult hippocampal stem cells during cocaine withdrawal: possible role of the cell fate regulator FADD

Abstract

The current study uses an extended access rat model of cocaine self-administration (5-h session per day, 14 days), which elicits several features manifested during the transition to human addiction, to study the neural adaptations associated with cocaine withdrawal. Given that the hippocampus is thought to have an important role in maintaining addictive behavior and appears to be especially relevant to mechanisms associated with withdrawal, this study attempted to understand how extended access to cocaine impacts the hippocampus at the cellular and molecular levels, and how these alterations change over the course of withdrawal (1, 14, and 28 days). Therefore, at the cellular level, we examined the effects of cocaine withdrawal on cell proliferation (Ki-67+ and NeuroD+ cells) in the DG. At the molecular level, we employed a 'discovery' approach with gene expression profiling in the DG to uncover novel molecules possibly implicated in the neural adaptations that take place during cocaine withdrawal. Our results suggest that decreased hippocampal cell proliferation might participate in the adaptations associated with drug removal and identifies 14 days as a critical time-point of cocaine withdrawal. At the 14-day time-point, gene expression profiling of the DG revealed the dysregulation of several genes associated with cell fate regulation, highlighting two new neurobiological correlates (Ascl-1 and Dnmt3b) that accompany cessation of drug exposure. Moreover, the results point to Fas-Associated protein with Death Domain (FADD), a molecular marker previously associated with the propensity to substance abuse and cocaine sensitization, as a key cell fate regulator during cocaine withdrawal. Identifying molecules that may have a role in the restructuring of the hippocampus following substance abuse provides a better understanding of the adaptations associated with cocaine withdrawal and identifies novel targets for therapeutic intervention.

Figure 1

Escalation of cocaine intake during LgA cocaine SA procedures. (a) Experimental design (see Materials and methods section). (b) Data represent mean±SEM amount of cocaine intake (0.5 mg/kg cocaine per infusion) over the entire daily sessions (5-h session per day of cocaine SA) for 14 days in adult male Sprague–Dawley rats (LgA, n=20). Rats progressively increased their daily cocaine intake starting at an average of 15 mg/kg on the first day of SA and finishing at an average intake of about 50 mg/kg on the final SA session day (day 1 vs day 14; ^***p<0.0001).

Figure 2

Decreased proliferation of adult hippocampal stem cells. (a, b) Quantitative analysis of Ki-67+ (a) and NeuroD+ (b) cells in the left hemisphere DG revealed a decrease in cell proliferation at Day 14 of withdrawal following LgA cocaine SA. ^**p<0.01, ^*p<0.05 when compared with their respective control (ND-Day 14). (c, d) Analysis split by anterior (−1.80 to −4.52 mm from Bregma) and posterior (−4.52 to −6.80 mm from Bregma) hippocampal demarcation. The posterior DG had more Ki-67+ and NeuroD+ cells than the anterior DG. ^***p<0.001, ^**p<0.01. Ki-67+ and NeuroD+ cells were decreased both in the anterior and posterior demarcation (Ki-67: ^*p<0.05; NeuroD: ^*p<0.05) in LgA-Day 14 when compared with ND-Day 14. (e, f) Quantitative analysis of Ki-67+ (e) and NeuroD+ (f) cells in relation to distance from Bregma in ND-Day 14 and LgA-Day 14 groups. (g, h) Representative IHC of Ki-67+ (g, brown labeling) and NeuroD+ (h, dark blue labeling) comparing ND vs LgA at Day 14. The bigger images were taken in a light microscope using a 20 × objective lens to illustrate the anatomy of the DG. For each image, the left-bottom corner shows a representative IHC image taken in a light microscope using a 63 × oil objective lens. Scale bar: 25 μm. The color reproduction of this figure is available at the Neuropsychopharmacology journal online.

Figure 3

Correlation analysis. (a) Scatterplot depicting a significant positive correlation between the number of Ki-67+ and Neuro+ cells in the DG of rats independently of their treatment. Each symbol represents a different rat. The solid line is the best fit of the correlation (r=0.76, n=37, p<0.0001). The dotted curves indicate the 95% confidence interval for the regression line. To note that this correlation is still there for each treatment group individually (ND groups: r=0.84, n=17, p<0.0001; LgA groups: r=0.60, n=20, p=0.005). (b) Scatterplot depicting a significant negative correlation between the number of Ki-67+ cells and the amount of cocaine intake (mg/kg) in the DG of LgA groups rats. Each symbol represents a different rat. The solid line is the best fit of the correlation (r=0.51, n=20, p=0.02). The dotted curves indicate the 95% confidence interval for the regression line. To note that there was no correlation when comparing the number of NeuroD+ cells with the amount of cocaine intake (r=0.03, n=20, p=0.91).

Figure 4

Possible role of the cell fate regulator FADD. (a) ISH analysis: data represents mean±SEM mRNA level of LgA-Day 14 rats when compared with the percentage of the ND-control group (100%) at 14 days of withdrawal (ND-Day 14) in the left hemisphere DG determined by ISH analysis (^*p=0.014). (b) ISH analysis: FADD mRNA across the anatomical level of the DG analyzed (−1.80 to −5.80 mm from Bregma) (^*p<0.05, ^***p<0.001 vs ND-Day 14). (c) ISH analysis: representative X-ray images of FADD mRNA for each treatment group at two levels of analysis (anterior DG: −2.30 mm and, posterior DG: −4.80 mm from Bregma) (d) WB analysis: data represent mean±SEM protein level (LgA-Day 14), and are expressed as percentage of the ND-control group (100%) at 14 days of withdrawal (ND-Day 14) in dissected right hippocampus. Student's t-test did not detect significant changes between groups. FADD: nonsignificant 19% increase, p=0.09. The figure also illustrates representative immunoblots of the corresponding proteins. β-Actin was used as a loading control. (e) Assay for apoptotic cell death. ND-Day 14 and LgA-Day 14 showed similar levels of basal 116 kDa PARP cleavage (∼85 kDa fragment) indicating similar rates of basal induction of apoptotic cell death in the right hemisphere hippocampus. The figure also illustrates a representative immunoblot of the corresponding protein.