Time-course of AMPAR redistribution during cocaine withdrawal
Data from seven behavioral trials (some reported previously; Boudreau and Wolf 2005
) were used for the current study. In each trial, rats received seven injections of saline or cocaine (15 mg/kg on treatment days 1 and 7; 30 mg/kg on days 2–6). Locomotor activity was measured on treatment days 1 and 7. As expected from prior studies using this regimen (Pierce et al. 1996
; Churchill et al. 1999
; Boudreau and Wolf 2005
; Boudreau et al. 2007
), about half of the cocaine-treated rats developed sensitization based on comparison of treatment days 1 and 7 for saline and cocaine groups (see Boudreau and Wolf 2005
for criteria). Thus, each trial yielded saline controls, sensitized rats and non-sensitized rats.
shows locomotor activity counts on treatment days 1 and 7 for rats destined for biochemical analysis on withdrawal day (WD) 1, WD7, or WD21. Within each group (saline, sensitized and non-sensitized), rats destined for analysis at different withdrawal times had equivalent responses on treatment days 1 and 7. Saline rats showed no change in activity between days 1 and 7, as expected ( left). Sensitized rats showed significantly higher activity on day 7 (, middle). Non-sensitized rats showed no change between treatment days 1 and 7 (, right). However, their initial response to cocaine on treatment day 1 was greater than the initial response of rats that developed sensitization, whereas their response on day 7 was lower (p
< 0.05 in both cases; comparison of all non-sensitized versus all sensitized rats). We did not measure stereotypy so we cannot rule out the possibility that the “non-sensitized” group failed to exhibit enhanced locomotion on treatment day 7 because they had developed stereotyped behaviors incompatible with locomotion. However, in preliminary studies with this regimen, we did not observe stereotyped behaviors that would preclude locomotion, and results of Sabeti et al. (2003
) argued against this explanation for failure of a subgroup of rats to develop locomotor sensitization to cocaine. Furthermore, the non-sensitized rats resembled saline controls more than sensitized rats in their biochemical profile (see subsequent sections and Discussion).
Fig. 1 Withdrawal from cocaine is accompanied by increased GluR1 S/I ratios in the NAc of sensitized rats. (a) Rats received seven daily injections of saline (SAL) or cocaine. Cocaine rats were divided into sensitized (SENS) and non-sensitized (NON) groups based (more ...)
NAc tissue was obtained from all rats and cross-linked with BS3
, a membrane impermeant cross-linking agent. BS3
cross-links cell surface proteins, increasing their apparent mass, while intracellular proteins remain unmodified, enabling surface (S) and intracellular (I) pools to be distinguished by SDS-PAGE and western blotting. The NAc from one rat yields enough cross-linked tissue for ~15 immunoblots. One aliquot from each rat was used to measure the GluR1 surface/intracellular ratio (S/I), while others were used to assess signaling pathways (see next section). GluR1 was studied because most AMPARs in the NAc contain this subunit (Boudreau et al. 2007
; Conrad et al. 2008
We found that the sensitized group exhibited significantly higher GluR1 S/I ratios than control rats on WD7 and WD21 but not WD1, indicating that AMPAR surface expression increases during the first week of withdrawal and then remains elevated for at least two more weeks (). Then we used a Spearman Rank Order test to determine if individual rats exhibited correlations between GluR1 S/I and the magnitude of behavioral sensitization (). On WD1, no positive correlations were found; in fact, nonsensitized rats yielded a significant negative correlation. On WD7, a positive correlation between GluR1 S/I and the magnitude of sensitization existed for all cocaine-exposed rats. When cocaine-exposed rats were divided into sensitized and non-sensitized groups, only sensitized rats showed a positive correlation. Similarly, on WD21, a positive correlation was observed for all cocaine-exposed rats and again, after division into sensitized and non-sensitized groups, only sensitized rats showed a positive correlation. Thus, withdrawal-dependent increases in AMPAR surface expression were observed only in rats that developed locomotor sensitization.
Table 1 Spearman rank order r and p-values for relationships between the magnitude of sensitization (Day 7/Day 1), GluR1 S/l ratio, pERK2/ERK2, pCaMKII/CaMKII, pCaMKII/total protein, CaMKII/total protein and overall PKA substrate phosphorylation in saline (SAL), (more ...)
We also compared S, I and total (S + I) GluR1 levels in NAc tissue from sensitized, non-sensitized and saline groups (data not shown, but results for some WD21 trials were reported previously; Boudreau and Wolf 2005
). In every group where GluR1 S/I increased, surface GluR1 also increased significantly, whereas intracellular levels tended to decrease, though not significantly. GluR1 total protein levels in sensitized rats were modestly but not significantly increased at all three withdrawal times (115–124% of control). Other studies using similar regimens have detected increases in total GluR1 of similar magnitude that achieved statistical significance (Churchill et al. 1999
; Ghasemzadeh et al. 2009
Next, signaling pathways (CaMKII, ERK and PKA) known to be important for hippocampal AMPAR trafficking (see Introduction) were examined in NAc total tissue homogenates from these same rats (tissue was cross-linked, but this does not affect detection of intracellular antigens).
AMPAR redistribution is paralleled by increased ERK2 phosphorylation
Ras-ERK signaling is implicated in AMPAR synaptic insertion in hippocampal neurons (Zhu et al. 2002
), so we examined ERK activation using phospho-specific antibodies. Cocaine withdrawal did not alter pERK1 or total ERK1 (data not shown). For ERK2, no group differences were found on WD1 (). However, on WD7 and WD21, when the GluR1 S/I ratio was increased in sensitized rats, pERK2/ERK2 was also increased in sensitized rats () whereas total ERK2 protein was unchanged ( ERK2 measurements for non-sensitized rats did not differ from saline controls. Thus, increased pERK2/ERK2 paralleled the increased AMPAR surface expression observed in sensitized rats on WD7 and WD21.
Fig. 2 ERK2 phosphorylation, like AMPAR surface expression, is increased in the NAc of sensitized rats (SENS) on WD7 and WD21 compared with non-sensitized (NON) and saline (SAL) rats. (a–c) PhosphoERK2 normalized to total ERK2 protein (pERK2/ERK2). (d–f) (more ...)
We evaluated correlations between pERK2/ERK2 and the GluR1 S/I ratio for individual rats on WD1, WD7 and WD21 (). A significant positive correlation was observed for saline controls on WD7 and WD21. This may reflect the normal role of ERK2 signaling in AMPAR synaptic incorporation as inferred from results of Zhu et al. (2002)
. Lack of correlation on WD1 may indicate perturbation of normal relationships shortly after discontinuing the mildly stressful experience of daily saline injections. Among cocaine groups, the only significant relationship was a positive correlation between pERK2/ERK2 and GluR1 S/I for all cocaine-exposed rats on WD7 that was not present when sensitized and non-sensitized groups were assessed separately, perhaps due to lower N
-values. Thus, while the increases in GluR1 S/I and pERK2/ERK2 occurred in parallel, the magnitude of these increases was not significantly correlated in sensitized rats, suggesting an indirect relationship between the two.
CaMKII phosphorylation is increased in cocaine-exposed rats on WD7
Activation of CaMKII induces AMPAR synaptic insertion during hippocampal LTP (Hayashi et al. 2000
), so we assessed CaMKII phosphorylation in our NAc aliquots. Sensitized rats on WD1 exhibited decreased pCaMKII/CaMKII () but no change in total CaMKII protein (). On WD7, pCaMKII and total CaMKII levels increased in both sensitized and non-sensitized rats (). Because both measures increased proportionally, there was no overall change in pCaMKII/CaMKII in either group (). By WD21, there were no significant between-group differences for any CaMKII parameter ().
Fig. 3 CaMKII protein and phosphorylation are increased after 7 days of withdrawal in the NAc of sensitized (SENS) and non-sensitized rats (NON) compared with controls (SAL). (a–c) PhosphoCaMKII normalized to total CaMKII protein (pCaMKII/CaMKII). (d–f) (more ...)
We also examined possible correlations for individual rats between CaMKII measurements and either the magnitude of sensitization or GluR1 S/I (). On WD1, pCaMKII/CaMKII was negatively correlated with the magnitude of sensitization for all cocaine-exposed rats but not saline controls, consistent with between-group results showing decreased pCaMKII/CaMKII in sensitized rats (). No relationship was found between pCaMKII/CaMKII and GluR1 S/I for any group. On WD7, no significant correlations were found between any CaMKII measure and the magnitude of sensitization or GluR1 S/I, despite between-group differences for some CaMKII measures on WD7 as described above (). Finally, on WD21, pCaMKII/CaMKII was negatively correlated with the GluR1 S/I ratio for all cocaine-exposed rats and sensitized rats, but not for non-sensitized rats.
In hippocampal neurons, Ras-ERK activation is downstream of CaMKII in the pathway leading to AMPAR synaptic insertion (Zhu et al. 2002
), so we also evaluated correlations between pCaMKII/CaMKII and pERK2/ERK2 for individual rats (). Although no statistically significant correlations were found, there was a trend towards a positive correlation in saline rats at all withdrawal times (p
= 0.09–0.22) and in sensitized rats on WD1 and WD7 (p
= 0.24 and 0.19, respectively).
In summary, a transient increase in CaMKII signaling may contribute to AMPAR up-regulation on WD7 but it is not sufficient, as CaMKII is activated in non-sensitized rats on WD7 and they do not develop AMPAR up-regulation.
Phosphorylation of PKA substrates increases in sensitized rats during late withdrawal
Protein kinase A signaling is implicated in AMPAR trafficking to the cell surface (Chao et al. 2002a
; Esteban et al. 2003
; Mangiavacchi and Wolf 2004
; Sun et al. 2005
; Gao et al. 2006
; Oh et al. 2006
; Man et al. 2007
). We assessed PKA indirectly by immunoprobing with an antibody recognizing the phosphorylated consensus site for PKA phosphorylation. Antibody specificity was confirmed in control experiments (see Materials and methods) including peptide competition experiments demonstrating that immunoreactivity detected with the PKA substrate antibody is eliminated by the PKA substrate peptide used to raise the antibody but not a PKC substrate peptide (). Under our conditions, nine major PKA substrate bands ranging from ~90 kDa to ~39 kDa were detected in NAc tissue using the phosphorylated PKA substrate antibody ().
We began by quantifying overall PKA substrate phosphorylation (sum of the nine bands) in NAc tissue from the same rats analyzed for other measures. In sensitized rats, overall PKA substrate phosphorylation was significantly decreased on WD1, unchanged on WD7, and significantly increased on WD21 compared with saline controls, indicating that it lagged temporally behind AMPAR up-regulation during withdrawal ().
Next, we evaluated correlations between overall PKA phosphorylation and other measures for individual rats. Results are shown in . No significant correlations between overall PKA substrate phosphorylation and GluR1 S/I were found for sensitized rats. However, on WD1 and WD7, there were trends towards an inverse relationship (r = –0.77 and –0.49, respectively; p = 0.1 for both days), which switched towards a positive relationship (r = 0.57, p = 0.1) on WD21. This shift, combined with a gradual increase in PKA substrate phosphorylation as withdrawal progresses (), is consistent with a possible role for PKA in maintaining AMPAR up-regulation in sensitized rats late in withdrawal.
The withdrawal-dependent increase in overall PKA phosphorylation () was paralleled by results obtained when each of the nine prevalent PKA substrate bands were analyzed individually for the sensitized group (). On WD1, phosphorylation of PKA substrates 66, 46, and 42 kDa was significantly decreased in sensitized rats and a trend in the same direction was observed for several other bands (). On WD7, phosphorylation of several PKA substrates appeared to be recovering in the direction of control levels (). On WD21, the phosphorylation of PKA substrates 90, 66, 54, 46, 42, and 39 kDa was significantly increased compared with saline controls (). Thus, we can identify specific substrate proteins that contribute to the gradual increase in overall PKA substrate phosphorylation that occurs in the NAc between WD1 and WD21 in sensitized rats (). No change in the phosphorylation of any of the nine prevalent PKA substrate bands was observed in non-sensitized rats compared with controls at any withdrawal time (data not shown).
Fig. 5 Phosphorylation of individual PKA substrates increases slowly over the course of withdrawal in the NAc of cocaine-sensitized rats. Panels a–i show data for nine prevalent PKA substrate bands. WD 1: n = 7; WD 7: n = 7; WD 21: n = 10. Data (mean (more ...)
Next, we tested possible correlations between each PKA substrate band and GluR1 S/I or the magnitude of sensitization (day 7/day 1). We restricted the analysis to WD21 because this is the only withdrawal time when sensitized rats showed overall elevation of PKA substrate phosphorylation (). Of the individual PKA substrates showing elevated phosphorylation in sensitized rats on WD21 (), only PKA substrate band 39 kDA exhibited positive correlations with the other measures. Phosphorylation of this substrate on WD21 correlated with GluR1 S/I in sensitized rats (r = 0.5, p < 0.05) and with both GluR1 S/I and the magnitude of sensitization in all cocaine-exposed rats (r = 0.5, p < 0.05 and r = 0.44, p < 0.05, respectively). These relationships were not present at earlier withdrawal times (data not shown).
We also tested possible correlations between individual PKA substrate phosphorylation and pERK2/ERK in sensitized rats on WD21, based on our observation that both measures were elevated in the sensitized group on WD21 ( and ). Only PKA substrate band 54 kDa showed a positive correlation (r = 0.69, p < 0.05). Finally, because pCaMKII/total protein and CaMKII/total protein increased on WD7 in sensitized and non-sensitized groups (), while PKA substrate phosphorylation was “in transition” at this withdrawal time ( and ), we evaluated possible correlations on WD7 between these CaMKII measures and PKA substrate phosphorylation. pCaMKII/total protein showed significant positive correlations with seven PKA substrates for individual cocaine-exposed rats on WD7 [substrates 90, 78, 74, 42, and 39 kDa (p < 0.01) and 66, 54, and 46 kDa (p < 0.05)]. For all of these bands, significant positive correlations (90, 78, 74, 42, 39 kDa; p < 0.05) or trends (66, 54, 46 kDa; p-values < 0.12) were also found when sensitized rats were assessed separately. In non-sensitized rats, only one significant correlation (39 kDa; p < 0.05) and one trend (42 kDa; p = 0.12) were found. Thus, while pCaMKII/total protein was increased in both sensitized and non-sensitized groups on WD7 (), only sensitized rats also exhibited correlations on WD7 between pCaMKII/total protein and PKA phosphorylation of multiple substrates (as well as overall PKA phosphorylation; see ), and only sensitized rats showed AMPAR up-regulation. Total CaMKII protein, also elevated in both sensitized and non-sensitized groups on WD7 (), was positively correlated with phosphorylation of certain substrates (90, 74, 42, 39 kDa; p < 0.05) for individual rats in the cocaine-exposed group on WD7. However, none of these correlations persisted when sensitized rats were analyzed separately (data not shown).
Proteomic identification of PKA substrate proteins of 90, 66, 54, and 39 kDa
We selected four substrate proteins for additional experiments (90, 66, 54 and 39 kDA) based on significant increases in their phosphorylation in sensitized rats on WD21 () and withdrawal-dependent correlations with other measures (see previous section). As a first step towards identifying these PKA substrates, we performed proteomic analyses on excised bands from colloidal Coomassie- and SYPRO Ruby-stained 1 dimensional SDS-PAGE gels with subsequent identification of bands by mass spectrometry. The starting material was NAc tissue obtained from two adult, drug-naïve rats. Bands to be excised were identified by aligning stained gels and western blots probed with antibody recognizing phosphorylated PKA substrates. First, we analyzed a gel loaded with 400 μg of NAc tissue (Run A). Electrospray ionization MS/MS (ESI-MSn) analyses of the excised and protease-cleaved bands at ~90, ~66, ~54, and ~39 kDa revealed several interesting high score hits (Run A; ). Of these, heat shock protein of 90 kDa (HSP90) α and HSP90 β were identified for the band excised at ~90 kDa, HSP of 70 kDa (HSP70) and Collapsin Response Mediator Protein-2 (CRMP-2) were identified for the band excised at ~66 kDa, α-tubulin was identified as the highest probability hit for the band excised at ~54 kDa band, and guanine nucleotide binding protein alpha-o (Goα) subunit 2 (Goα2) was identified as highest probability hit for the band excised at ~39 kDa (). Next, we performed a more focused, higher resolution and sensitive ESI-MSn analysis of a second sample run on a one-dimensional gel, this time loading ~150 μg of NAc protein (Run B). Of the high probability hits for the ~66 kDa band from Run A, CRMP-2 was again identified while HSP70 was not (Run B; ). Again, we found α-tubulin and Goα2 to be the highest probability hits for the 54 kDa and the 39 kDa excised bands, respectively ().
Mass spectrometry results for bands excised at 90, 66, 54, and 39 kDa corresponding to the molecular weights of individual PKA substrate bands (PKA Sub.) from Coomassie stained one-dimensional gels
Bioinformatics examination of the amino acid sequences with Net PhosK v.1.0 (http://www.cbs.dtu.dk/services/netPhosK
) or Phosphosite v.2.0 (http://www.phosphosite.org
) revealed that HSP90, CRMP-2, α-tubulin, and Goα2
each contained putative PKA phosphorylation site(s), supporting the hypothesis that these proteins contributed to immunoreactivity detected with the PKA substrate antibody. Another aliquot of the same tissue used for runs A and B was separated by two-dimensional gel electrophoresis to further spatially resolve the proteins and assess the population of phosphorylated species, and then immunoblotted with PKA substrate antibody. This revealed that there were multiple phosphorylation sites with different isoelectric points, consistent with differential and combinatorial phosphorylation sites in the 90, 66, 54, and 39 kDa ranges (Fig. S1
) migrating along the same mass. Each individual spot was not of high enough abundance to identify the site of phosphorylation. Furthermore, phosposerine and phosphothreonine are labile (e.g. beta-elimination) so different mass spectrometric strategies need to be employed and require higher abundance of protein.
CRMP-2 was identified as a component of the post-synaptic density by mass spectrometry (Peng et al. 2004
) and is of interest as a potential mediator of structural plasticity (see Discussion). Goα2
is another interesting candidate because it transduces signals from DA receptors (Neve et al. 2004
). To determine if their localization is consistent with participation in AMPAR plasticity in NAc neurons, we performed immunocytochemical experiments in primary cultures prepared from postnatal rat NAc neurons. Both CRMP-2 and Goα2
are expressed in the processes and cell bodies of NAc medium spiny neurons (Fig. S2