Article Figures & Data
Figures
- Figure 1.
DGL protein levels in the NAc core are unchanged after prolonged withdrawal from extended-access cocaine (Coc) self-administration. A, Two cohorts of rats underwent extended-access self-administration of cocaine or saline (6 h/d for 10 d), destined for preparation of NAc core homogenates after WD40 (WD > 40; top) or NAc synaptoneurosomes on WD40 (bottom). Shown are the number of infusions (left graphs) or number of active hole (AH) and inactive hole (IH) nose pokes (right graphs) on the 10 d of self-administration training. N values are given in the figure. B, DGL protein levels do not differ between saline and cocaine groups based on immunoblot analysis of NAc homogenates (top) or synaptoneurosomes (bottom). Data are expressed as the percentage of controls (n = 7–9 rats/group; mean ± SEM). Representative lanes from immunoblots (cropped) are shown for a saline rat (left) and a cocaine rat (right). Arrows indicate bands analyzed, and lines indicate the location of the molecular weight marker. Saline and cocaine groups were compared with unpaired t tests (n.s.).
- Figure 2.
Associations between components of the 2-AG signalosome in the NAc core are unchanged after prolonged withdrawal from extended-access cocaine (Coc) self-administration. A, Rats underwent extended-access self-administration of cocaine or saline (6 h/d for 10 d), destined for the preparation of NAc core homogenates on WD55. Shown are the number of infusions (left graph) or the number of active hole (AH) and inactive hole (IH) nose pokes (right graph) during the 10 d of self-administration training. N values are given in the figure. B, Coimmunoprecipitation experiments assessing the physical associations between DGL and mGlu5 or Homer2 in NAc tissue obtained on WD55 (also see Extended Data Figure 2-1). DGL was immunoprecipitated from NAc core homogenates, and the bound fraction was immunoblotted for mGlu5 or Homer2. Data are expressed as the percentage of saline (Sal) controls (n = 9–10 rats/group; mean ± SEM). Representative lanes from immunoblots (cropped) are shown for a saline rat (left) and a cocaine rat (right). Arrows indicate bands analyzed, and lines indicate the location of the molecular weight marker. IB, Immunoblot; IP, Immunoprecipitation. Saline and cocaine groups were compared with unpaired t tests (n.s.).
- Figure 3.
The association of CaMKII with DGL is increased in the NAc core after prolonged withdrawal from extended-access cocaine (Coc) self-administration. Coimmunoprecipitation experiments assessed the physical associations between DGL and phosphorylated or total CaMKIIα and CaMKIIβ in NAc core homogenates prepared on WD55 (same rats for which behavioral data are shown in Fig. 2). Also see Extended Data Fig. 3-1. Data are expressed as the percentage of the saline (Sal) control group (n = 6–10 rats/group; mean ± SEM; unpaired t tests, *p < 0.05, #p = 0.0501 vs saline). Representative lanes from immunoblots (cropped) are shown for a saline rat (left) and a cocaine rat (right). Arrows indicate bands analyzed, and lines indicate the location of the molecular weight marker. IB, Immunoblot.
- Figure 4.
Reduction of DGL activity in the NAc after prolonged withdrawal from extended-access cocaine (Coc) self-administration. A, Training. Rats self-administered saline or cocaine for 6 h/d for 10 d (n = 9 rats/group). Shown are the number of infusions (left graph) or number of active hole (AH) and inactive hole (IH) nose pokes (right graph). B, Seeking tests on WD1 and WD40. During the 30 min test, AH nose pokes led to contingent presentation of the 20 s light cue previously paired with cocaine infusion, but no cocaine. Shown are AH and IH nose pokes during the seeking test (mean ± SEM; paired t test, **p < 0.01 vs WD1). C, DGL activity. Five days after the WD40 seeking test, bilateral NAc punches comprised primarily of NAc core were snap frozen. Fluorescence resonance energy transfer assay of DGL activity was then conducted and results expressed as ΔRFU/minute. The cocaine group shows reduced DGL activity (n = 7–9 rats/group; mean ± SEM; unpaired t test, *p < 0.05 vs saline). D, Linear regression evaluating the relationship between DGL activity in NAc core of cocaine animals versus the magnitude of incubation of craving expressed as WD40/WD1 AH nose pokes (n = 7 rats; R = −0.692, p = 0.085).
- Figure 5.
DGL activity in the NAc after prolonged withdrawal from extended-access cocaine (Coc) self-administration does not correlate with the magnitude of incubation of craving. A, Training. Rats self-administered cocaine for 6 h/d for 10 d (n = 9 rats). Shown are the number of infusions (left graph), and the number of active hole (AH) and inactive hole (IH) nose pokes (right graph). B, Seeking tests on WD1 and WD42. Shown are AH and IH nose pokes during the 30 min seeking test (mean ± SEM; paired t test, **p < 0.01 vs WD1). C, Five days after the WD42 seeking test, bilateral NAc punches comprised primarily of NAc core were snap frozen and DGL activity was determined using fluorescent resonance energy transfer (Extended Data Fig. 5-1). D, Linear regression evaluating DGL activity versus the magnitude of incubation of craving expressed as WD42/WD1 AH nose pokes. No significant correlation between DGL activity and incubation was found (n = 9 rats; R = 0.393; n.s.). E, Combined linear regression evaluating DGL activity versus the magnitude of incubation of craving from both cocaine cohorts (shown in Fig. 4A and Fig. 5A). From comparison of Fig. 4C and Fig. 5C, it is apparent that absolute RFU per minute values for the two cohorts differed substantially (the cohorts were run over a year apart). To enable the data from the cohorts to be combined, mean values (ΔRFU/minute) were calculated for each cohort and individual rat values (ΔRFU/minute) were expressed as percentage of the cohort mean (Fig. 5A cohort in black, n = 7; Fig. 4A cohort in gray, n = 9; R = 0.199, n.s.). RFU, Relative Fluorescence Units.
Extended Data
Figure 2-1
Assessment of associations between DGL, mGlu5, and Homer1b/c in the NAc of drug-naive animals. A, Coimmunoprecipitation experiments assessing the association between DGL and mGlu5. Left, Results of an experiment in which DGL was immunoprecipitated from NAc core homogenates, followed by immunoblotting for mGlu5 in the starting material (SM; homogenate), Bound (B) or immunoprecipitated fraction, and unbound (UB) fraction. mGlu5 is present in all fractions. Right, Results after immunoprecipitating mGlu5 from the same starting material and immunoblotting for DGL. Although DGL is more abundant in starting material and unbound fractions, it is detectable in the bound fraction. B, C, Coimmunoprecipitation experiments assessing the association between DGL and Homer1b/c. Although Homer1b/c is present in the starting material (B), it is not detected in the bound fraction after immunoprecipitation of DGL (C); the arrow in C shows the molecular weight at which the Homer1b/c band should have been observed. IB, Immunoblot. Download Figure 2-1, TIF file.
Figure 3-1
Assessment of DGL phosphorylation in the NAc after prolonged withdrawal from extended-access cocaine (Coc) self-administration. Rats underwent extended-access self-administration of cocaine or saline (6 h/d for 10 d), destined for preparation of NAc core homogenates on WD55 (Fig. 2, behavioral data). DGL was immunoprecipitated from NAc core homogenates (six saline rats, nine cocaine rats), and the bound fraction was immunoblotted with an antibody that recognizes phosphorylated serine residues (p-Serine). No group difference was found (t(13) = 2.54, p = 0.897). However, as noted in the main text, these data are not conclusive. Because we did not originally plan to assess the phosphorylation state of DGL using this tissue, some of the buffers used in the immunoprecipitation protocol did not contain phosphatase inhibitors. Thus, DGL may have been dephosphorylated during the lengthy immunoprecipitation protocol. IB, Immunoblot. Download Figure 3-1, TIF file.
Figure 5-1
DGL activity measurements in NAc core of cocaine rats and in two control groups. Rats self-administered cocaine (Fig. 5A) and received cue-induced seeking tests on WD1 and WD42 (Fig. 5B). Five days after the WD42 seeking test, bilateral NAc punches composed primarily of NAc core were snap frozen and DGL activity was determined using fluorescent resonance energy transfer. Triplicate values for each of nine cocaine rats are shown, along with duplicates for two control groups: cerebellum tissue (CB) and cerebellum tissue plus the DGL inhibitor DO34 (CB + DO34). Download Figure 5-1, TIF file.
In this issue
