Molecular changes in the medial prefrontal cortex and nucleus accumbens are associated with blocking the behavioral sensitization to cocaine

Previous studies have demonstrated that cocaine-induced behavioral sensitization is associated with persistent functional and structural alterations in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc); however, the molecular mechanisms underlying these changes have not been elucidated. In this study, the behavioral sensitization to cocaine was established in Sprague Dawley rats and was measured by locomotion and behavioral rating. The brain tissue homogenization was used for measuring the level of brain-derived neurotrophic factor (BDNF), the expression and activity of integrin-linked kinase (ILK), level of protein kinase B (Akt) phosphorylation at serine 473 and threonine 308, and the expression of p75NTR, TrkA, and TrkB protein. The Results showed that cocaine sensitization was associated with increased BDNF, ILK activity, phospho-Akt Ser473, p75NTR, and TrkB protein levels in the mPFC and NAc core. The combination of pergolide and ondansetron normalized not only behavioral sensitization, but also the increases in these molecular markers. Dual immunofluoresence staining showed that ILK expression is co-distributed with p75NTR and TrkA expression in both the mPFC and NAc core. Results suggested that the BDNF-TrkA/p75NTR-ILK-Akt signaling pathway may be active in cocaine sensitization and associated neural plasticity in the mPFC and NAc core.


Materials and Methods
Animals and Drugs. Male Sprague-Dawley rats (SLAC LABORATORY ANIMAL Inc., Shanghai, China), initially weighing 150-200 g, were housed in pairs in the animal facility of Central South University on a 12-h light/dark cycle for 1 week prior to the experiments. Rats were then housed individually after initial vehicle or cocaine injection. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and Chinese legislation on the use and care of laboratory animals. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques. All experimental protocol were approved by the Institutional Committee of Central South University Cocaine HCl (Qinghai Pharmaceutical Factory, China), ondansetron hydrochloride dihydrate (GlaxoSmithkline, Research Triangle Park, NC), and pergolide (Sigma, St. Louis, MO) were prepared as described 28 .
Experimental Groups and Behavioral Measurements. Animals were treated as previously described 28 . Briefly, rats received 25 mg/kg/day cocaine (s.c.) for 5 consecutive days, followed by withdrawal for 9 days to establish long-term cocaine sensitization, or rats received saline injections plus 9 days of withdrawal. Starting on day 10 of withdrawal, half of the rats that received cocaine (C-P/O) or saline (S-P/O) injections were given 0.1 mg/kg pergolide, followed by 0.2 mg/kg ondansetron (s.c.) 3.5 h later for 5 consecutive days. The other half of the rats in the cocaine (C-D/S) and saline groups (S-D/S) received parallel vehicle injections for 5 consecutive days. All animals were then subjected to 9 days of withdrawal, followed by a challenge injection of 7.5 mg/kg cocaine (i.p.) (Fig. 1). Animals were then immediately returned to their home cages. The locomotion and behavioral rating scores were monitored over the next 60 min as previously described 28 . The baseline of locomotion in all animals was measured 1 day before cocaine injection. The behavioral score was given as 1 = asleep, 2 = almost asleep, 3 = dystonia, 4 = inactive, 5 = in-place oral behavior, 6 = grooming, 7 = normal-active movement, 8 = hyperactive, 9 = slow-patterned movement, 10 = fast-patterned movement, and 11 = stereotypy. Brain Dissection and Protein Measurement. Rats were euthanized and decapitated 24 h after the acute cocaine challenge. Brains were rapidly removed and sectioned for 1 mm coronal sections for medial prefrontal cortex (mPFC), caudate putamen (CPU), nucleus accumbens (NAc) core, NAc shell, and amygdala. Especially, mPFC was taken at + 3.2 to + 2.2 mm; CPU, NAc core, and NAc shell at + 2.0 to + 1.0 mm; and amygdala at − 2.3 to − 3.3 mm 28 . The tissue below the commissure was sectioned with the most ventral part designated as the NAc shell and the dorsal portion as the NAc core. Samples were stored at − 80 o C until protein extraction. BDNF Protein. BDNF levels in brain tissue lysates were measured using the BDNF Emax ImmunoAssay System kit (Promega, Madison, WI) according to the manufacturer's instructions. The brain tissue lysates were diluted 1:4 with sample buffer provided with the kit. The BDNF standard curve was run for each plate (linear range: 7.8-500 pg/ml BDNF) and samples from a given brain area were determined in single assays.
Western Blots. Western blots were performed as previously described 28 . The primary antibody [anti-ILK 1:1000; anti-phospho-Akt (Ser 473 )1:1000; anti-phospho-Akt (Thr 308 ) 1:1000; anti-Akt 1:1000] and peroxidase-labeled secondary antibody (1:2000 dilution) were purchased from Cell Signaling Technology (Berverly, MA). The rabbit anti-TrkA, anti-TrkB antibodies and anti-p75 NTR antibody were purchased from Millipore (1:1000 dilution, Temecula, CA). The blot was developed with chemiluminescent substrate (Santa Cruz Biotechnology, Santa Cruz, CA). To control for loading efficiency, the blots were stripped and re-probed with α -tubulin antibody (1:1000 dilution; Abcam, Cambridge, MA) and expression levels of total ILK and phospho-Akt proteins were normalized to that of α -tubulin. ILK Activity Assay. ILK activity assay was conducted as previously described 29 , but with some modifications. Briefly, tissue lysates (100 μ g) were incubated with 2 μ g anti-ILK antibody (Millipore, Temecula, CA ) or IgG as a negative control overnight at 4 °C, followed by addition of 50 μ l protein A/G PLUS agarose beads (Santa Cruz Biotechnology) and continual incubation for 2 h at 4 °C. The immune complex was isolated by centrifugation, washed 5× with 1-ml homogenate buffer and then 2x subsequently with kinase reaction buffer. Kinase activity was determined by incubating the immunoprecipitated complex with 1 μ g of inactive Akt-GST agarose (Millipore) and 200 μ M of ATP in reaction buffer for 1 h at 30 °C. ILK activity was determined by Western blot of Akt-GST phosphorylation using the site-specific anti-phospho-Akt (Ser 473 ) antibody (Cell Signaling).

Data analyses.
The data were presented as means and standard errors of the mean and were analyzed using the Statistical Package for the Social Sciences, Version 20.0. The ambulation and biochemical results were analyzed using one-way ANOVA, while repeated measures ANOVA was used for the behavioral rating scores. The post-hoc analyses were performed using Bonferroni corrected pair-wise comparisons. A p < 0.05 was considered statistically significant.

Results
BDNF Changes in the mPFC and NAc Core Parallel Behavioral Sensitization to Cocaine. ANOVA revealed significant effects of treatments on ambulation [F (3,34) = 6.724, p < 0.001]. The C-D/S animals had significantly higher ambulation in response to cocaine challenge than the S-D/S (p < 0.001), S-P/O (p < 0.01), and C-P/O (p < 0.015) groups (Fig. 2a). The behavioral rating scores (Fig. 2b To determine whether these long-term changes in the brain could be blocked, BDNF levels in the mPFC, NAc core, NAc shell, CPU, and amygdala were measured using ELISA. No significant treatment effects were detected in the NAc shell, CPU, or amygdala (Table 1). In contrast, ANOVA revealed significant treatment effects on BDNF levels in the mPFC [F (3,31) = 19.197, p < 0.001] and NAc core [F (3,31) = 45.460, p < 0.001] (Fig. 2c,d). BDNF levels were significantly increased in the mPFC (p < 0.001) and NAc core (p < 0.001) in the sensitized (C-D/S) rats compared to rats in all other groups. Importantly, pergolide/ondansetron treatment (C-P/O) significantly normalized BDNF levels in the mPFC and NAc core. These findings suggest that: 1) cocaine sensitization selectively leads to long-term increases (> 23 days after last cocaine injection, C-D/S group) in concentrations of BDNF in the mPFC and NAc core; and 2) combined pergolide/ondansetron treatment not only blocks the behavioral sensitization, but also normalizes BDNF levels in these two brain areas. 30 . Whether ILK levels parallel BDNF concentrations in the mPFC and NAc core has not been previously reported. Western blot showed that no changes were observed in ILK protein expression levels in the CPU, NAc shell, or amygdala, following establishment of cocaine sensitization (Table 1). In contrast, significant group differences were observed in the mPFC [F (3,31) = 11.155, p < 0.001] and NAc core [F (3,31) = 29.961, p < 0.001] (Fig. 3a,b). In the mPFC, ILK levels were significantly higher in the C-D/S group than in the S-D/S (p < 0.001), S-P/O (p < 0.001), and C-P/O groups (p < 0.001). Pergolide/ondansetron treatment (C-P/O) significantly normalized ILK levels in cocaine sensitized rats. Similar to the finding in the mPFC, ILK levels in the NAc core were significantly higher in C-D/S animals than in the other three groups (ps < 0.001) (Fig. 3b). We further investigated whether the changes in ILK expression resulted in parallel changes in ILK activity. Cocaine sensitization increased ILK activity (Fig. 3c, p < 0.05) which correlated with ILK expression (Fig. 3d) in the mPFC. This suggested that ILK activity is related to the ILK protein level.

Cocaine Sensitization Increases ILK Protein and Phospho-Ser 473 Akt Levels in the mPFC and NAc Core. A previous study demonstrated that ILK is involved in cocaine sensitization
Total Akt levels were not significantly altered among all animals in any tested brain areas (Table 1). In contrast, phospho-Ser 473 -Akt levels (normalized to total Akt protein) exhibited significant treatment effects in the mPFC and NAc core (Fig. 4a,b). One-way ANOVA revealed that levels of phospho-Ser 473 Akt in the mPFC [F (3,31) = 15.545, p < 0.001] and NAc core [F (3,31) = 17.615, p < 0.001] were significantly different among treatment groups, but not in the CPU, NAc shell, or amygdala (Table 1). Bonferroni tests demonstrated that the relative levels of Akt Ser 473 phosphorylation were increased in the mPFC of C-D/S rats compared to those in the S-D/S (p < 0.001) and C-P/O groups (p < 0.001), but not in the S-P/O group (Fig. 4a). In the NAc core, post-hoc analyses revealed that the level of Akt Ser 473 phosphorylation was significantly higher in the C-D/S group than in all other groups (p < 0.001) (Fig. 4b). These changes in the levels of phospho-Ser 473 Akt and ILK in the mPFC and NAc core parallel changes in the levels of BDNF protein in the same brain regions. In contrast, phosph-Thr 308 Akt levels were not significantly different between groups in both the mPFC (Fig. 4c) and NAc core (Fig. 4d).

Cocaine Sensitization Increases TrkB and p75 NTR Protein Levels in the mPFC and NAc Core.
It is well known that BDNF binds to the TrkB receptor and activates several signal pathways, including PI3K-Akt 11 . A previous study demonstrated that BDNF-mediated activation of ILK-Akt signaling requires co-expression of TrkA and p75 NTR receptors 12 . TrkA, TrkB, and p75 NTR expression were measured in brain tissues. No significant differences in TrkA, TrkB, and p75 NTR expression were observed in the CPU, NAc shell, or amygdala following establishment of cocaine sensitization (Table 1). In contrast, significant group differences in TrkB levels were observed in the mPFC [F (3,31) = 11.155, p < 0.001] and NAc core [F (3,31) = 19.961, p < 0.001] (Fig. 5A,B). In the mPFC, TrkB level was elevated in the C-D/S group relative to those in the S-D/S, S-P/O, and C-P/O groups (p < 0.001). The C-P/O group was not statistically different from either the S-D/S or S-P/O group (Fig. 5a). Similar to its levels in the mPFC, TrkB levels in the NAc core for C-D/S animals were enhanced compared to those in the other three groups (p < 0.001), which were not different from one another (Fig. 5b). In contrast, no significant group differences or significant differences in TrkA levels in the mPFC (Fig. 5c) and NAc core (Fig. 5d) between groups (p > 0.05) were observed. Significant group differences in p75 NTR levels were observed in the mPFC [F (3,31) = 15.343, p < 0.001] and NAc core [F (3,31) = 17.163, p < 0.001] (Fig. 5e,f) NAc core, p75 NTR level was elevated in the C-D/S group relative to those in the S-D/S, S-P/O, and C-P/O groups (p < 0.001). The C-P/O group was not statistically different from either the S-D/S or S-P/O group (Fig. 5e,f).

TrkA and p75 NTR co-locate with ILK Expression. Previous studies have demonstrated that ILK
is localized to neuronal cell bodies and dendrites in various brain regions 21 . TrkA and p75 NTR are co-expressed on the cortical and striatal neurons 31,32 . A study in cell culture also demonstrated that BDNF can activate ILK-Akt through stimulating TrkA/p75 NTR heteroreceptor 12 . We therefore investigated whether the expression of TrkA and p75 NTR is co-located with ILK in the mPFC and NAc core, regions that showed changes in BDNF/ILK expression. The dual immunofluoresence staining revealed that TrkA and p75 NTR expression are co-distributed with ILK expression in the mPFC and NAc core (Fig. 6). This suggests that ILK might be activated by BDNF through stimulation of TrkA/p75 NTR heteroreceptor.

Discussion
The present study has shown that long-term behavioral sensitization to cocaine is accompanied by increases in BDNF, TrkB, p75NTR, ILK, and phospho-Ser 473 Akt protein levels in the mPFC and NAc core -two brain areas that have been demonstrated to be intimately involved in cocaine abuse. Moreover, combined pergolide/ondansetron treatment blocked either the behavioral alterations or the increases in the above signaling molecules. Although cocaine sensitization is known to produce long-lasting changes in behavior and molecules related to synaptic plasticity 19,33,34 , our results first suggested an involvement of the BDNF-TrkA/p75 NTR -ILK-Akt signaling pathway in cocaine-induced long-lasting behavioral alterations. Importantly, both the sensitization-associated behaviors and molecules could be blocked by the clinically available drugs. A previous study in PC-12 cells demonstrated that BDNF-mediated activation of ILK-Akt signaling requires co-expression of TrkA and p75 NTR receptors 12 . In vivo studies have shown the co-expression of TrkA and p75 NTR receptors on the cortical and striatal neurons 31,32 . TrkA and p75 NTR receptors were thought to often exist on the same neurons, coordinating and modulating neuronal neurotrophin responses by suppressing or enhancing each other's actions 32,35 or by reciprocally modulating the receptor affinity states in the TrkA and p75 NTR heteroreceptor complex 36 . However, whether the cocaine sensitization is also associated with the activation of BDNF-ILK-Akt signaling, particularly, through the activation of TrkA and p75 NTR heteroreceptor remains inadequately addressed. Consistent with previous studies stated above, this study demonstrated that p75 NTR and TrkA receptors were co-located in the NAc core and mPFC, and were also co-located with ILK expression in these two brain areas. However, Western blot demonstrated that p75 NTR , but not TrkA expression was upregulated in the mPFC and NAc core, paralleling the increases in ILK expression and Akt Ser 473 phosphorylation. Therefore, we hypothesized that: 1) BDNF-dependent activation of ILK-Akt signaling in the mPFC and NAc core may be mediated through TrkA/p75 NTR heteroreceptors. It requires only the coordinate increase in p75 NTR activation. In contrast, TrkA may only be required to be co-expressed as a coordinator; and 2) enhanced p75 NTR activation and subsequent ILK-Akt signaling may play a significant role in cocaine behavioral sensitization and its reversal by combined pergolide/ondansetron treatment. It is well established that BDNF specifically binds to the tyrosine kinase B (TrkB) receptor and activates various signaling cascades, which include PI3K-dependent activation of Akt where the Thr 308 and Ser 473 residues become phosphorylated 7,37 . In this study, consistent with increases in BDNF concentrations, cocaine sensitization parallels increases in the levels of TrkB and phospho-Ser 473 Akt, but not phospho-Thr 308 in the mPFC and NAc core. Among signaling molecules that regulate Akt phosphorylation, ILK is one of the upstream kinases that can phosphorylate Akt at the Ser 473 residue 38 . The present study demonstrated that changes in ILK protein levels parallel those in the levels of BDNF and phospho-Ser 473 Akt following establishment of sensitization. In addition, the change in ILK protein levels parallel the change in ILK enzymatic activity in vivo. A previous study demonstrated that established cocaine sensitization is associated with opposite changes in the PI3K activity levels in the mPFC and NAc core, but pergolide/ondansetron treatment fails to block either of these changes 19 . These divergent alterations in the status of PI3K are quite different from the parallel changes observed in BDNF-ILK-Akt Ser 473 signaling and behavioral sensitization in the present study. Taken together, these observations suggest that: 1) BDNF is an initiator of ILK-Akt signaling which may not need the corresponding changes in TrkB-PI3K signaling to become activated. In contrast, it may only require constitutive PI3K activity to become activated although pergolide/ondansetron treatment blocked TrkB levels in rats that were administered cocaine; and 2) dysregulation of BDNF-TrkA/p75 NTR -ILK-Akt signaling may play a significant role in long-term maintenance of cocaine sensitization. While the discrepancy between PI3K-and ILK-mediated signaling events may be attributed to a number of different factors, the present study suggests that these two events are mediated by different neurotrophin receptors and thus are independent of each other although constitutive PI3K activity may be required. Other PI3K-independent pathways could also participate in BDNF-Akt signaling during cocaine sensitization and its reversal. These transduction pathways may include MAPK; however, in this case, Akt activation is primarily achieved through Thr 308 phosphorylation 39 . Finally, the increased Akt signaling may also be due to an as yet-to-be-identified non-BDNF-dependent signaling molecule. For instance, NMDA or AMPA receptor stimulation also leads to phosphorylation of Akt 40,41 . Long-term cocaine sensitization is associated with brain region-dependent alterations in phosphorylation of the NR2B and GluR1 subunits of the respective NMDA and AMPA receptors, and these changes are blocked by pergolide/ondansetron treatment 28 . However, this study provided no further evidence on how BDNF was activated and how the BENF-TrkA/p75 NTR -ILK-Akt signaling was involved in the long-term behavioral sensitization to cocaine.
Previous studies have demonstrated that addictive drugs induce cAMP increase via activation of dopamine receptors and calcium entry via activation of glutamate NMDA receptors. These events, in turn, activate numerous intracellular signaling cascades 7,42,43 . Among them, activated CREB contributes to regulation of genes important to the neuroplastic changes, including regulation of BDNF mRNA transcription, translation, and translocation to dendrites 9,44-46 . BDNF is secreted locally near active synapses and binds to Trk receptors located on presynaptic and postsynaptic sites. The activated BDNF/ Trks signaling can also phosphorylate AMPA receptors and subsequently activates the CREB and may result in progressive BDNF gene expression and release 44,47 . There are many ways to interfere with signaling involved in BDNF transcription and translation, which subsequently affects BDNF levels and the sensitization-associated synaptic plasticity. Here, pergolide is a DA agonist and its behavioral effects can last for 3-4 hrs in animals 48 . Previous studies showed that pergolide alone was not sufficient to consistently block the sensitization-associated behavioral and molecular changes 28,49 . However, pergolide can induce an 'acute DA withdrawal state' 28 . 5-HT 3 terminal receptors were thought to mediate local stimulatory actions of 5-HT on DA release in the mPFC, striatum, and NAc 28 . Ondansetron is a 5-HT 3 antagonist. A previous study suggested that pergolide may evoke a methamphetamine associated memory and that ondansetron can disrupt its reconsolidation 50 . Thus, modulations of the DA-5-HT 3 interactions in the mPFC and NAc core may form a basis for blocking the sensitization-associated behaviors and BDNF/ ILK/Akt changes by a pergolide/ondansetron treatment regimen.
In addition to changes in signal transduction, chronic abuse of drugs (e.g., cocaine) can produce persistent structural alterations in the cytoskeleton. These changes include increases in dendritic branching and density of dendritic spine on medium spiny neurons in the NAc and pyramidal cells in the PFC [51][52][53] . BDNF stimulation can also lead to similar structural changes 54,55 . Furthermore, ILK can regulate integrin-dependent neurite outgrowth in N1E-115 neuroblastoma cells 23 and NGF-stimulated differentiation of PC-12 cells and dorsal root ganglion neurons 21 . These observations suggest that increased BDNF levels may be associated with the morphological plasticity observed in long-term psychostimulant (cocaine) sensitization through the BDNF-ILK signaling cascade. Precise relationships of this neurobiological process with functional and morphological changes in behavioral sensitization await further elucidation.
In conclusion, this study suggests that the BDNF-TrkA/p75 NTR -ILK-Akt signaling pathway may be active in cocaine sensitization and could be associated with neural plasticity in the mPFC and NAc core.