Cocaine-induced dendritic spine formation in D1 and D2 dopamine receptor-containing medium spiny neurons in nucleus accumbens

Abstract

Psychostimulant-induced alteration of dendritic spines on dopaminoceptive neurons in nucleus accumbens (NAcc) has been hypothesized as an adaptive neuronal response that is linked to long-lasting addictive behaviors. NAcc is largely composed of two distinct subpopulations of medium-sized spiny neurons expressing high levels of either dopamine D1 or D2 receptors. In the present study, we analyzed dendritic spine density after chronic cocaine treatment in distinct D1 or D2 receptor-containing medium-sized spiny neurons in NAcc. These studies made use of transgenic mice that expressed EGFP under the control of either the D1 or D2 receptor promoter (Drd1-EGFP or Drd2-EGFP). After 28 days of cocaine treatment and 2 days of withdrawal, spine density increased in both Drd1-EGFP- and Drd2-EGFP-positive neurons. However, the increase in spine density was maintained only in Drd1-EGFP-positive neurons 30 days after drug withdrawal. Notably, increased DeltaFosB expression also was observed in Drd1-EGFP- and Drd2-EGFP-positive neurons after 2 days of drug withdrawal but only in Drd1-EGFP-positive neurons after 30 days of drug withdrawal. These results suggest that the increased spine density observed after chronic cocaine treatment is stable only in D1-receptor-containing neurons and that DeltaFosB expression is associated with the formation and/or the maintenance of dendritic spines in D1 as well as D2 receptor-containing neurons in NAcc.

Fig. 1.

Analysis of MSNs in Drd1-EGFP and Drd2-EGFP mice. (a and b) Fixed brain slices from NAcc of Drd1-EGFP (a) or Drd2-EGFP (b) BAC transgenic mice were immunostained for GFP and NeuN (as a general neuronal marker). The merged images show, in yellow, colocalization of GFP and NeuN. Of neurons in NAcc of Drd1-EGFP mice, 58% expressed GFP, whereas 48% of neurons in NAcc of Drd2-EGFP mice expressed GFP. The numbers of GFP-positive and NeuN-positive neurons were counted independently; all GFP-positive neurons were also NeuN-positive. (c) Visualization of DiI in a single MSN in NAcc. The region of NAcc analyzed in Lower is indicated in the schematic in Upper (red circle) (Bregma, 1.34 mm). Twenty pictures were taken at different z levels (0.5- to 1-μm intervals); the images were then stacked and flattened. (d) Examples of distal dendrites visualized with DiI and used for analysis of spines. (Scale bars: a–c, 50 μm; d, 10 μm.)

Fig. 2.

Analysis of dendritic spines in Drd1-EGFP and Drd2-EGFP mice. Neurons in NAcc of either Drd1-EGFP mice (a) or Drd2-EGFP mice (b) were first labeled with DiI (red) and then subjected to immunohistochemistry using an anti-GFP antibody (EGFP, green). Only MSNs were labeled with DiI. Images of DiI staining and GFP staining were overlaid (Merge). (a Upper) The dashed circles show an example of double labeling of a DiI-positive and Drd1-EGFP-positive cell. (a Lower) The dashed squares show a lack of double labeling of cells in slices from Drd1-EGFP mice. (b Upper) The dashed circles show an example of double labeling of a DiI-positive and Drd2-EGFP-positive cell. (b Lower) The dashed squares show a lack of double labeling of cells in slices from Drd2-EGFP mice. The rightmost images in a Upper and b Upper show examples of DiI-stained distal dendrites in Drd1-EGFP or Drd2-EGFP mice, respectively. (Scale bars: 10 μm.) The examples shown were from saline-treated mice.

Fig. 3.

Chronic cocaine-induced increases in spine density in Drd1-EGFP- or Drd2-EGFP-positive MSNs in NAcc. (a and b) Drd1-EGFP (a) or Drd2-EGFP (b) mice were treated with saline (Sal) or cocaine (Coc, 30 mg/kg) for 4 weeks. Mouse brains 2WD or 30WD were processed for DiI labeling and immunohistochemistry as shown in Fig. 2. All spine-like protrusions on distal dendrites were included in the analysis. Data are expressed as the number of spines per 10 μm of dendritic length (mean ± SEM); ∗∗∗, P < 0.001 versus saline treated group, Kolmogorov–Smirnov test. (c and d) Cumulative frequency plots showing the distribution of spine density from individual neurons that were analyzed in a and b. One to two dendrites from one neuron in 10–15 animals per group were analyzed. The total numbers of dendrites analyzed were as follows. For D1: saline, 49 (2WD) and 38 (30WD); cocaine, 74 (2WD) and 75 (30WD). For D2: saline, 50 (2WD) and 43 (30WD); cocaine, 79 (2WD) and 89 (30WD).

Fig. 4.

Chronic cocaine induces ΔFosB expression in Drd1-EGFP- or Drd2-EGFP-positive MSNs in NAcc. Drd1-EGFP (a, c, and e) or Drd2-EGFP (b, d, and f) mice were treated with saline or chronic cocaine as described in Fig. 3. 2WD (c and d) or 30WD (e and f), the expression of GFP (green) for the identification of Drd1-EGFP- or Drd2-EGFP-positive MSNs and ΔFosB (red) was analyzed by immunohistochemistry. The localization of NeuN (purple) was also analyzed to show the position of neuronal nuclei. NeuN, GFP, and ΔFosB images were merged to examine their coexpression (Merge). Photomicrographs are representative of results from multiple brain sections obtained from three to four animals in each treatment group. Quantitative analysis is shown in Table 1.