- Email: [email protected]
VEGF regulates antidepressant effects of lamotrigine
European Neuropsychopharmacology (2012) 22, 424–430
www.elsevier.com/locate/euroneuro
VEGF regulates antidepressant effects of lamotrigine Rongju Sun a , Nanxin Li b,⁎, Tanshi Li a,⁎⁎ a b
Department of Emergency, General Hospital of PLA, Beijing 100853, China Department of Psychology, Yale University, New Haven, CT 06520, USA
Received 18 July 2011; received in revised form 29 August 2011; accepted 29 September 2011
KEYWORDS Antidepressant; VEGF; CUS; FST; Lamotrigine; NSF
Abstract The anticonvulsant drug lamotrigine has been shown to produce strong antidepressant effects in the treatment of bipolar disorder patients. Our previous studies have demonstrated that brain derived neurotrophic factor (BDNF) signaling plays an important role in regulating its behavioral actions in several rodent models of depression. The current study extends earlier work on BDNF and explores the role of another important neurotrophin vascular endothelial growth factor (VEGF) in regulating the antidepressant actions of lamotrigine. The results showed that chronic administration of 30 mg/kg lamotrigine (14 days) normalized the down-regulated frontal and hippocampal VEGF protein expression as well as the behavioral deficits induced by chronic unpredictable stress. In addition, pharmacological inhibition of VEGF signaling by infusion of SU5416, a selective Flk-1 receptor inhibitor, blocks the antidepressant effects of lamotrigine in all behavioral paradigms. Taken together, this study provides further evidence that VEGF is also an essential regulator for the antidepressant effects of lamotrigine. © 2011 Elsevier B.V. and ECNP. All rights reserved.
1. Introduction Bipolar disorder is a severe neuropsychiatric disease that affects about 2% of the population. Although there are many available mood stabilizers, most of them precipitate the onset of the manic episode and induce rapid cycling of manic and depressive phases (Bourin and Prica, 2007). An ⁎ Correspondence to: N. Li, Department of Psychology, Yale University, New Haven, CT 06520, USA. Tel.: + 1 203 8156200. ⁎⁎ Correspondence to: T. Li, Department of Emergency, General Hospital of PLA, Beijing 100853, China. Tel.: +86 10 66936224. E-mail addresses: [email protected] (N. Li), [email protected] (T. Li).
antiepileptic drug lamotrigine has revealed some antidepressant-like effects in patients with bipolar disorders without precipitating mania (Bowden et al., 1999; Calabrese et al., 1999, 2001). Previous rodent studies with lamotrigine using the forced swim test (FST) (Ali et al., 2003; Bourin et al., 2005; Consoni et al., 2006; Li et al., 2010; Prica et al., 2008) have generated some controversial results, which can largely be explained by the dose responses. Another shortcoming of FST is that it is responsive to acute antidepressant treatments, whereas chronic administration of antidepressants is required for the therapeutic effects (Berton and Nestler, 2006). In contrast to FST, chronic unpredictable stress (CUS) is considered one of the better rodent models of depression, which mimics the
0924-977X/$ - see front matter © 2011 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2011.09.010
VEGF regulates antidepressant effects of lamotrigine anhedonia symptom of depression and is only responsive to chronic antidepressant medications. Using this model, our previous work demonstrated that chronic treatment of lamotrigine (30 mg/kg, 21 days) reversed the anhedonic-like symptoms in the sucrose preference test and anxiogenic-like behaviors in the novelty suppressed feeding test (Li et al., 2011), thus had more convincingly evaluated the behavior actions of lamotrigine in rodent animal models of depression. The neurobiological mechanisms underlying the antidepressant effects of lamotrigine also remain largely obscure. The neurotrophic hypothesis of depression states that stress or depression decreases the expression of certain neurotrophic factors in several cortical and limbic brain regions and antidepressant treatments reverses or even blocks this neurotrophic factor deficit to reverse the neuronal atrophies (Duman, 2004). For example, previous studies by many labs including ours have showed that sub-chronic or chronic treatment of lamotrigine increased brain derived neurotrophic factor (BDNF) expression levels in the frontal cortex and hippocampus (Chang et al., 2009; Li et al., 2010). Although much emphasis have been put on BDNF, recent studies have begun to highlight another neurotrophin vascular endothelial growth factor (VEGF) in the neural mechanism of stress/depression and antidepressant (Greene et al., 2009; Heine et al., 2005; Jin et al., 2002; Storkebaum et al., 2004; Warner-Schmidt and Duman, 2007, 2008). In particular, VEGF is shown to mediate the neurogenic and behavioral actions of common antidepressants including fluoxetine and desipramine (Segi-Nishida et al., 2008; Warner-Schmidt and Duman, 2007). In the current study, we aim to directly test if VEGF signaling is also required for the antidepressant effects of lamotrigine.
2. Experimental procedures 2.1. Animals Male Sprague–Dawley rats (Beijing Laboratory Animal Center) weighing 175–200 g were housed in two and maintained under standard conditions with a 12 h light/dark cycle and ad libitum access to food and water. Two experiments were performed in this study (n = 6 to 8 for each group). The first experiment has two variables: stress condition (CUS and non-stressed, see below) and drug treatment (lamotrigine and vehicle). The second experiment, in which all rats were surgerized and subjected to the CUS procedure, also has two variables: infusion treatment (SU5416 and DMSO) and drug treatment (lamotrigine and vehicle). Behavioral tests were performed from 10 AM to 5 PM concurrent with stated housing conditions. The experiments were carried out in accordance with the guidelines of the Beijing Laboratory Animal Center and the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23).
2.2. Drug treatment Lamotrigine (Sigma, St Louis, MO) was dissolved in a mixture of 0.5% carboxymethylcellulose (Sigma, St Louis, MO), 0.4% Tween 80 (American Bioanalytical, Natick, MA), 0.9% benzylic acid (Sigma, St Louis, MO) and saline, which served as the control vehicle in all behavioral and molecular assays. For CUS/behavior experiments, animals were randomly assigned into different drug treatment groups, receiving vehicle or 30 mg/kg daily administration of lamotrigine. The doses used were based on previous studies (Bourin et al., 2005; Consoni et al., 2006; Li et al., 2010, 2011; Prica, et al.,
425 2008). All drugs were administered intraperitoneally (i.p.) at 1.0 ml/kg. Lamotrigine treatment was initiated one week after onset of CUS experiment and lasted for 14 days. The VEGF-Flk-1 selective antagonist SU5416 (4 mM, Sigma, St Louis, MO) or the vehicle (DMSO, Sigma, St Louis, MO) were delivered in a 1 μl volume at the rate of 0.25 μl i.c.v. with a cannula (26GA, PlasticOne, Roanoke, VA) protruding 0.5 mm beyond the guide cannula (PlasticOne, Roanoke, VA) on days 14, 16, 18, and 20. The dose and time course for SU5416 was based on previous studies (Greene et al., 2009; WarnerSchmidt and Duman, 2007).
2.3. Stererotaxic surgeries Six days before the initiation of the CUS paradigm, rats were anesthetized with Nembutal (i.p. 55 mg/kg, Butler Schein, Dublin, OH) and a single guide cannula was stereotaxically placed into the lateral ventricle (coordinates relative to bregma: −0.9 mm AP, −1.5 mm ML, and −3.3 mm DV from dura) and held in place with aluminum screws and dental cement. Postoperative care consisted in peri-surgerical administration of carprofen (5 mg/kg, Butler Schein, Dublin, OH) and triple antibiotic.
2.4. Chronic unpredictable stress (CUS) procedure In CUS, animals were exposed to a variable sequence of mild and unpredictable stressors (Willner, 2005). Our CUS procedure was successfully used to produce depressive-like behavioral changes (Greene et al., 2009; Warner-Schmidt and Duman, 2007). A total of 10 different stressors are used (two stressors per day for 21 days). The stressors used included rotation on a shaker, placement in a 4 °C ambient, lights off for 3 h (10 AM to 1 PM), lights on overnight, strobe light overnight, aversive odor, 45° tilted cages, food and water deprivation, crowding housing and isolation housing. The same CUS procedure was used for both lamotrigine experiments with or without surgery. Control animals were handled every other day and animal weights were monitored.
2.5. Sucrose preference test (SPT) The SPT consisted of a 48 h exposition (days 20 and 21) of rats to a palatable sucrose solution (1%; Sigma, St. Louis, MO), followed by 4 h of water deprivation on day 22 and a 1 h exposure to two identical bottles, one filled with sucrose solution and the other with water. This procedure was adapted from previous studies and has been used previously in our lab (Warner-Schmidt and Duman, 2007). Sucrose and water consumption were determined by measuring the change in the volume of fluid consumed. Sucrose preference was defined as the ratio of the sucrose versus the total consumption (sucrose and water) during the 1 h exposure.
2.6. Novelty-suppressed feeding test (NSFT) The NSFT was performed as previously described (Greene et al., 2009). After the SPT the rats were food-deprived for 12 h and on day 23 placed in an open field (76.5 cm × 76.5 cm × 40 cm, Plexiglas) with a small amount of food in the center. Animals were allowed to explore the open field for 8 min. The sessions were filmed by a camcorder from above and the latency to feed, specifically, the time it took for the animal to approach and take its first bite of the food, was recorded offline by a stopwatch in seconds. Home cage food intake was also measured as a control.
2.7. VEGF protein detection For the first lamotrigine experiment (without surgery) on day 24 the animals were killed by decapitation, brains were removed and both
426 halves of the frontal cortex and the hippocampal tissue were dissected for protein assays. Tissues were kept on dry ice and stored at − 80 °C. After homogenizing by sonication, VGEF protein level of each sample was quantified by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN) following manufacturer's protocol. The intra- and inter-assay variation coefficients (CV%) of the VEGF ELISA assay were 3.7% and 4.6%, respectively. The average% of recovery of rat VEGF spiked was 99%. VGEF protein level was normalized to the total protein level determined by BCA analysis (Pierce, Rockford, IL). Results were expressed as pictogram of VGEF protein/milligram of total protein.
2.8. Statistical analysis All analyses were performed using SPSS 13.0 software and data were reported as mean ± S.E.M. Analysis of variance (ANOVA) followed by Fisher's post hoc test was conducted to compare group differences. The level of statistical significance was set at p b 0.05.
3. Results 3.1. Chronic lamotrigine treatment restores CUS-induced decrease of VEGF expression Chronic treatment of lamotrigine elevated frontal and hippocampal VEGF protein expression levels in both naïve and CUStreated animals (Fig. 1). The effect of stress was significant in both FC (F(1, 25) = 9.66, p b 0.01) and HC (F(1, 25) = 6.16, p b 0.05), indicating CUS significantly reduced VEGF expression in these two brain regions. The effect of drug was also significant in both FC (F(1, 25) = 11.51, p b 0.01) and HC (F(1, 20) = 11.33, p b 0.01). The interaction between stress and drug was not significant in either FC or HC (FC, p = 0.30; HC, p = 0.75). Further analysis indicated that in CUS-treated rats receiving lamotrigine showed a significant increase of VEGF expression as compared with the CUS group receiving vehicle (FC and HC, both p b 0.05), to a level comparable to non-stressed (NS) control rats (FC, p = 0.84; HC, p = 0.55). Lamotrigine also increased VEGF expression in non-stressed rats (FC and HC, both p b 0.05). The levels of total Flk-1 receptor remained comparable between different treatment groups (see supplemental materials).
R. Sun et al.
3.2. Chronic lamotrigine treatment ameliorates CUS-induced behavioral deficits Consistent with previous findings (Li et al., 2011), chronic treatment of lamotrigine produced robust antidepressant in SPT and NSFT (Fig. 2). For SPT (Fig. 2A), the interaction between stress condition and drug treatment was significant (F(1, 25) = 5.38, p b 0.05). The effect of stress was significant (F(1, 20) = 6.32, p b 0.05), indicating CUS produced potent anhedonia effects. The effect of drug was also significant (F(1, 20) = 7.36, p b 0.05). Further analysis indicated that in CUS-treated rats receiving lamotrigine showed a significant increase in sucrose preference compared with the CUS group receiving vehicle control (p b 0.01), to a level comparable to non-stressed (NS) control rats (p = 0.72). In addition, lamotrigine did not affect sucrose preference in non-stressed control rats (p = 0.76). There was no significant difference in total fluid consumption for the test (data not shown). For NSFT (Fig. 2B), the interaction between stress condition and drug treatment was significant (F(1, 25) = 4.63, p b 0.05). The effect of stress was significant (F(1, 25) = 5.48, p b 0.05), indicating CUS produced anxiogenic effects in rats. The effect of drug was also significant (F(1, 25) = 9.87, p b 0.01). Further analysis indicated that in CUS-treated rats receiving lamotrigine showed a significant decrease in latency to feed as compared with the CUS group receiving vehicle control (pb 0.01), to a level comparable to non-stressed (NS) control rats (p = 0.65). In addition, lamotrigine did not affect latency to feed in non-stressed control rats (p = 0.55). There was no significant difference in home cage food consumption (data not shown).
3.3. Blockade of VEGF-Flk-1 signaling abolishes the antidepressant effects of lamotrigine Blockaded of VEGF-Flk-1 signaling by inhibitor SU5416 completely abolished the antidepressant effects of lamotrigine in SPT and NSFT (Fig. 3). For SPT (Fig. 3A), the interaction between infusion and injection treatment was significant (F(1, 22) = 11.32, p b 0.01). The effect of infusion was significant (F(1, 22) = 5.64, p b 0.05). The effect of injection was also
Figure 1 Chronic lamotrigine treatment increased VEGF protein expression in FC (A) and HC (B). CUS + Lamotrigine rats showed higher FC and HC VEGF expressions to than CUS + Vehicle rats. There was no difference between CUS + Lamotrigine and nonstressed + Vehicle rats. Non-stressed + Lamotrigine rats also showed increased VEGF level than non-stressed + Vehicle rats. Results were expressed as mean + S.E.M., *, p b 0.05; #, non-significant, as compared with vehicle treated controls. n = 6 to 8 for each group. NS denotes non-stressed, CUS denotes chronic unpredictable stress, Veh denotes vehicle treatment, and Lam denotes lamotrigine treatment.
VEGF regulates antidepressant effects of lamotrigine
427
Figure 2 Chronic lamotrigine treatment produced robust antidepressant effects in SPT (A) and NSFT (B). CUS + Lamotrigine rats displayed higher sucrose preference and reduced latency to feed than CUS + Vehicle rats. There was no difference of sucrose preference and latency to feed between CUS + Lamotrigine and non-stressed + Vehicle rats. Results were expressed as mean + S.E.M., **, p b 0.01; #, non-significant, as compared with vehicle treated controls. n = 6 to 8 for each group. NS denotes non-stressed, CUS denotes chronic unpredictable stress, Veh denotes vehicle treatment, and Lam denotes lamotrigine treatment.
significant (F(1, 20) = 9.69, p b 0.01). Further analysis indicated that consistent with the last experiment, CUS-treated rats receiving lamotrigine showed a significant increase in sucrose preference compared with the CUS group receiving vehicle control (p b 0.01). This antidepressant effect of lamotrigine was completely blocked by infusion of the VEGF-Flk-1 antagonist: the CUS + lamotrigine + SU5416 group was not significantly different from the CUS + vehicle + DMSO or CUS + vehicle + SU5416 groups (p = 0.62, p = 0.86). There was no significant difference in total fluid consumption for the 1 h test (data not shown). For NSFT (Fig. 3B), the interaction between infusion and injection treatment was significant (F(1, 22) = 5.53, p b 0.05). The effect of infusion was significant (F(1, 22) = 6.77, p b 0.05). The effect of injection was also significant (F(1, 22) = 5.53, p b 0.05). Further analysis indicated that similar to last experiment, CUS-treated rats receiving lamotrigine showed a significant decrease of latency to feed as compared with the CUS group receiving vehicle control (p b 0.01). This antidepressant effect of lamotrigine was completely blocked by infusion of the VEGF-Flk-1 antagonist: the CUS + lamotrigine + SU5416 group was not significantly different from the CUS + vehicle + DMSO or CUS + vehicle + SU5416 groups (p = 0.76, p = 0.70). There was no
significant difference in home cage food consumption (data not shown).
4. Discussion The current study corroborates our previous studies and demonstrates that the mood stabilizer lamotrigine produces antidepressant effects in chronic unpredictable stress model of depression. Chronic lamotrigine treatment also up-regulates frontal and hippocampal VEGF expression levels in both nonstressed and stressed animals and completely reversed stressinduced VEGF deficits. Furthermore, pharmacological inhibition of VEGF-Flk-1 signaling completely abolishes antidepressant actions of lamotrigine. The dose of lamotrigine largely determines its therapeutic effects. In consistent with our recent studies (Li et al., 2010, 2011), we have used a relatively high dose (30 mg/kg) dose for the chronic administration. Previous studies reveal that lamotrigine can only decrease immobility in FST at high (10 mg/kg and above) but not lower doses (1 to 5 mg/kg) (Ali et al., 2003; Bourin et al., 2005; Li et al., 2010;
Figure 3 Blockade of VEGF-Flk-1 signaling abolished antidepressant actions of lamotrigine in SPT (A) and NSFT (B). CUS + DMSO + Lamotrigine rats displayed higher sucrose preference and reduced latency to feed than CUS + DMSO + Vehicle rats. There was no difference of su crose preference and latency to feed and immobility between CUS+ SU5416 + Lamotrigine and CUS+ SU5416 + Vehicle as well as CUS + DMSO + Vehicle rats. Results were expressed as mean + S.E.M., **, p b 0.01; #, non-significant, as compared with vehicle treated controls. n = 6 to 8 for each group. NS denotes non-stressed, CUS denotes chronic unpredictable stress, Veh denotes vehicle treatment, and Lam denotes lamotrigine treatment.
428 Prica et al., 2008). The 30 mg/kg dose used in the present study is well tolerated and does not cause any locomotor abnormalities (Li et al., 2010). The two rodent models currently used are only responsive to chronic antidepressant and thus better reflect the clinical therapeutic responses than behavioral despair models including FST. CUS is widely considered a better stress model since rodents develop a range of fundamental behavioral and neurochemical changes that resemble core symptoms of depression (Willner, 2005). One of the most evident abnormalities is reduced motivation for incentive stimuli, clinically known as anhedonia. Novelty suppressed feeding test is a model of anxiety and is commonly used in depression studies due to the high co-morbidity of these two diseases (Schmidt and Duman, 2007). Consistent with our previous report (Li et al., 2011), CUS produces severe anhedonic- and anxiogeniclike in rats, both of which are completely ameliorated by chronic lamotrigine treatment. Lamotrigine neither alters the behavior in non-stressed animals nor affects total fluid consumption or home-cage food intake, indicating the effects observed in the SPT and NSFT are specific to its antidepressant actions rather than its effects on metabolism. Therefore, together with recent findings by our and other laboratories, the current findings provide additional evidence on the preclinical antidepressant effects of lamotrigine. Most of previous studies on the neurobiological mechanism of lamotrigine have focused on BDNF and consistently demonstrated that chronic treatment of lamotrigine markedly increases BDNF mRNA and protein expression in several cortical and limbic regions including the frontal cortex and hippocampus (Li et al., 2011; Rao and Rapoport, 2009). The current study, for the first time, shows that lamotrigine can also activate another important neutrophic factor VEGF in the brain. Originally discovered as an endothelial survival marker, VEGF has recently shown strong neurogenic and neuroprotective effects and modulates synaptic activities and many hippocampal-dependent functions (Jin et al., 2002; McCloskey et al., 2005; Newton et al., 2003; Schanzer et al., 2004; Storkebaum et al., 2004). VGEF binds to two tyrosine receptors Flk-1 and Flt-1, of which the former is mainly expressed in the brain, in particularly the endothelial cells and neural progenitor cells in the HC (Warner-Schmidt and Duman, 2008). In particular, the up-regulation of VEGF-Flk-1 signaling is required for the behavioral and neurogenic effects of common antidepressants (Greene et al., 2009; WarnerSchmidt and Duman, 2007). The present study demonstrates that similar to fluoxetine and electroconvulsive shock (Warner-Schmidt and Duman, 2007), and chronic lamotrigine treatment can increase VEGF protein expression in both FC and HC, two important regions in the neural circuitry of depression. VEGF gene contains CRE and its expression can be stimulated by the activation of CREB (Greene et al., 2009). Previous studies have demonstrated that a variety of mood stabilizers, including lithium and valproate acid, can robustly increase phospho-CREB expression in the brain (Einat et al., 2003). It is reasonable to predict that lamotrigine may also regulate the expression of VEGF via activation of pCREB, though future studies were needed to confirm this hypothesis. Additional studies using double-label florescent in situ hybridization (FISH, with markers such as NeuN and GFAP) are needed to localize the source of VEGF release (neuron or glia) triggered by lamotrigine and to examine whether VEGF mRNA levels were
R. Sun et al. regulated or not. Furthermore, a recent study showed that veratrine, a sodium channel opener, can block the antidepressant effects of lamotrigine in FST (Prica et al., 2008). It will be interesting to study if there is any neurobiological connection among sodium channel, pCREB, and VEGF underlying the therapeutic effects of lamotrigine. More importantly, lamotrigine completely reverses the stressed-induced VEGF deficits to comparable levels of non-stressed controls. The neurotrophic hypothesis of depression postulates that stress/depression down-regulates the neurotrophic functions, which lead to structure and functional deficiencies in several key regions including FC and HC. In contrast, antidepressant medications produce therapeutic effects via reversing aforementioned neurotrophic changes and exerting neuroprotective effects (Duman, 2004). Chronic stress can decrease FC and HC VEGF levels, which can be alleviated by chronic antidepressant treatment (Greene et al., 2009; Heine et al., 2005; Warner-Schmidt and Duman, 2007). A recent study showed that chronic continuous exposure to corticosterone (CORT, a natural glucocorticoid) significantly reduced Flk-11 protein levels both in cultured cortical neurons and mouse frontal cortex (Howell et al., 2011). In the current study, we did not find any significant alteration of total Flk-1 proteins in frontal cortex or hippocampus. We reason that CUS and CORT are two different kinds of stress and it is possible that instead of regulating the total expression levels, CUS may regulate the activity of this receptor. Unfortunately all phosphor-Flk-1 antibodies we have attempted to use failed to generate consistent and reliable results and thus it remained an open question to be explored. Previous studies indicate that increasing VEGF signaling may a common pathway underlying the neurobiological mechanisms of various antidepressants, including lamotrigine. To directly test this hypothesis, we pharmacologically block the VEGF signaling in the brain by infusing Flk-1 receptor antagonist SU5416 and then investigate whether the infusion affects the behavioral effects of lamotrigine. VEGF preferentially binds to Flk-1 than Flt-1 receptor in the brain. Blockade of VEGF-Flk-1 signaling by intracerebroventricular infusion of the selective inhibitor SU5416 abolishes the antidepressant effects of fluoxetine and desipramine in several rodent models of depression, including CUS/SPT, NSFT, and FST (Greene et al., 2009; Warner-Schmidt and Duman, 2007). In the current study we find that SU5416 can also block the behavioral effects of lamotrigine in SPT and NSFT, indicating that VEGF-Flk-1 signaling indeed mediates the antidepressant actions of lamotrigine. Using a pharmacological inhibition approach, we have previously demonstrated that BDNF-TrkB signaling is also required for the therapeutic effects of lamotrigine in rodent models of depression (Li et al., 2011). It raises an interesting question on whether BDNF or VEGF (or both) are important for the antidepressant actions of lamotrigine. Previous studies have demonstrated that similar to lamotrigine, the therapeutic effects of many antidepressants, including the selective serotonin reuptake inhibitor fluoxetine, can activate both BDNF and VEGF signaling pathways in the brain to produce antidepressant effects (Greene et al., 2009; Pilar-Cuellar et al., 2011). Together with the current findings, we postulate that both pathways are necessary for the antidepressant actions of lamotrigine and neither pathway by itself is sufficient to
VEGF regulates antidepressant effects of lamotrigine mediate such actions. Future studies utilizing a combination of pharmacological inhibition and conditional knockout mice (since constitutive homozygous BDNF and VEGF knockout are both lethal) are needed to further address this issue. In addition, the specificity and potential cross-talk between different neurotrophic factor receptor antagonists warrant further investigation. For example, the effects of SU5416 on BDNF and TrkB and the effects of K252a on VEGF and Flk-1 should be extensively studied to help support the wide application of these inhibitors. There are a few limitations in this study. With the current drug delivery approach, it is difficult to determine in which part of brain is essentially involved in this signaling pathway. The current results suggest that FC and HC are good candidates for future studies with local microinjection to determine the regional specificity. In addition, although SU5416 is generally considered a reliably selective inhibitor of Flk1 receptor, like another commonly used inhibitor SU1498, the specificity of pharmacological inhibitors is still far from optimal. Other approaches, including genetic-mutant mice, should be used to more rigorously address the role of VEGF signaling in regulating antidepressant effects of lamotrigine. Taken together, the present study demonstrates that chronic treatment of lamotrigine increases FC and HC VEGF expressions and ameliorates CUS-induced behavioral deficits. Blockade of VEGF-Flk-1 signaling abolishes its therapeutic effects. The current findings expand our understanding of the potential neurobiological mechanisms of antidepressant actions of lamotrigine.
Role of the funding source Funding for this study was provided by National Natural Science Foundation of China and American Psychological Association; none of these grant sources had any further role in study design, in the collection, analysis and interpretation of data, in the writing of the report and in the decision to submit the paper for publication.
Contributors Authors TL and NL designed the study and wrote the protocol. Author RS and NL managed the literature searches and analyses. Author RS and NL undertook the statistical analysis, and authors TL and NL wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.
Conflict of interest All authors of this manuscript declare no conflict of interest.
Acknowledgments The current study was funded by grants from National Natural Science Foundation of China (30700869, 30840039) and American Psychological Association.
Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.euroneuro.2011.09.010.
429
References Ali, A., Pillai, K.K., Pal, S.N., 2003. Effects of folic acid and lamotrigine therapy in some rodent models of epilepsy and behaviour. J. Pharm. Pharmacol. 55, 387–391. Berton, O., Nestler, E.J., 2006. New approaches to antidepressant drug discovery: beyond monoamines. Nat. Rev. Neurosci. 7, 137–151. Bourin, M., Prica, C., 2007. The role of mood stabilisers in the treatment of the depressive facet of bipolar disorders. Neurosci. Biobehav. Rev. 31, 963–975. Bourin, M., Masse, F., Hascoet, M., 2005. Evidence for the activity of lamotrigine at 5-HT(1A) receptors in the mouse forced swimming test. J. Psychiatry Neurosci. 30, 275–282. Bowden, C.L., Mitchell, P., Suppes, T., 1999. Lamotrigine in the treatment of bipolar depression. Eur. Neuropsychopharmacol. 9 (Suppl. 4), S113–S117. Calabrese, J.R., Bowden, C.L., Sachs, G.S., Ascher, J.A., Monaghan, E., Rudd, G.D., 1999. A double-blind placebo-controlled study of lamotrigine monotherapy in outpatients with bipolar I depression. Lamictal 602 study group. J. Clin. Psychiatry 60, 79–88. Calabrese, J.R., Shelton, M.D., Rapport, D.J., Kujawa, M., Kimmel, S.E., Caban, S., 2001. Current research on rapid cycling bipolar disorder and its treatment. J. Affect. Disord. 67, 241–255. Chang, Y.C., Rapoport, S.I., Rao, J.S., 2009. Chronic administration of mood stabilizers upregulates BDNF and bcl-2 expression levels in rat frontal cortex. Neurochem. Res. 34, 536–541. Consoni, F.T., Vital, M.A., Andreatini, R., 2006. Dual monoamine modulation for the antidepressant-like effect of lamotrigine in the modified forced swimming test. Eur. Neuropsychopharmacol. 16, 451–458. Duman, R.S., 2004. Role of neurotrophic factors in the etiology and treatment of mood disorders. Neuromolecular Med. 5, 11–25. Einat, H., Manji, H.K., Gould, T.D., Du, J., Chen, G., 2003. Possible involvement of the ERK signaling cascade in bipolar disorder: behavioral leads from the study of mutant mice. Drug News Perspect. 16, 453–463. Greene, J., Banasr, M., Lee, B., Warner-Schmidt, J., Duman, R.S., 2009. Vascular endothelial growth factor signaling is required for the behavioral actions of antidepressant treatment: pharmacological and cellular characterization. Neuropsychopharmacology 34, 2459–2468. Heine, V.M., Zareno, J., Maslam, S., Joels, M., Lucassen, P.J., 2005. Chronic stress in the adult dentate gyrus reduces cell proliferation near the vasculature and VEGF and Flk-1 protein expression. Eur. J. Neurosci. 21, 1304–1314. Howell, K.R., Kutiyanawalla, A., Pillai, A., 2011. Long-term continuous corticosterone treatment decreases VEGF receptor-2 expression in frontal cortex. PLoS One 6, e20198. Jin, K., Zhu, Y., Sun, Y., Mao, X.O., Xie, L., Greenberg, D.A., 2002. Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc. Natl. Acad. Sci. U. S. A. 99, 11946–11950. Li, N., He, X., Qi, X., Zhang, Y., He, S., 2010. The mood stabilizer lamotrigine produces antidepressant behavioral effects in rats: role of brain-derived neurotrophic factor. J. Psychopharmacol. 23, 1772–1778. Li, N., He, X., Zhang, Y., Qi, X., Li, H., Zhu, X., He, S., 2011. Brainderived neurotrophic factor signalling mediates antidepressant effects of lamotrigine. Int. J. Neuropsychopharmacol. 1–8. McCloskey, D.P., Croll, S.D., Scharfman, H.E., 2005. Depression of synaptic transmission by vascular endothelial growth factor in adult rat hippocampus and evidence for increased efficacy after chronic seizures. J. Neurosci. 25, 8889–8897. Newton, S.S., Collier, E.F., Hunsberger, J., Adams, D., Terwilliger, R., Selvanayagam, E., Duman, R.S., 2003. Gene profile of electroconvulsive seizures: induction of neurotrophic and angiogenic factors. J. Neurosci. 23, 10841–10851.
430 Pilar-Cuellar, F., Vidal, R., Pazos, A., 2011. Subchronic treatment with fluoxetine and the 5-HT(2A) antagonist ketanserin upregulates hippocampal BDNF and beta-catenin in parallel with antidepressant-like effect. Br. J. Pharmacol. doi:10.1111/j.14765381.2011.01516.x (Electronic publication ahead of print). Prica, C., Hascoet, M., Bourin, M., 2008. Antidepressant-like effect of lamotrigine is reversed by veratrine: a possible role of sodium channels in bipolar depression. Behav. Brain Res. 191, 49–54. Rao, J.S., Rapoport, S.I., 2009. Mood-stabilizers target the brain arachidonic acid cascade. Curr. Mol. Pharmacol. 2, 207–214. Schanzer, A., Wachs, F.P., Wilhelm, D., Acker, T., Cooper-Kuhn, C., Beck, H., Winkler, J., Aigner, L., Plate, K.H., Kuhn, H.G., 2004. Direct stimulation of adult neural stem cells in vitro and neurogenesis in vivo by vascular endothelial growth factor. Brain Pathol. 14, 237–248. Schmidt, H.D., Duman, R.S., 2007. The role of neurotrophic factors in adult hippocampal neurogenesis, antidepressant treatments
R. Sun et al. and animal models of depressive-like behavior. Behav. Pharmacol. 18, 391–418. Segi-Nishida, E., Warner-Schmidt, J.L., Duman, R.S., 2008. Electroconvulsive seizure and VEGF increase the proliferation of neural stem-like cells in rat hippocampus. Proc. Natl. Acad. Sci. U. S. A. 105, 11352–11357. Storkebaum, E., Lambrechts, D., Carmeliet, P., 2004. VEGF: once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays 26, 943–954. Warner-Schmidt, J.L., Duman, R.S., 2007. VEGF is an essential mediator of the neurogenic and behavioral actions of antidepressants. Proc. Natl. Acad. Sci. U. S. A. 104, 4647–4652. Warner-Schmidt, J.L., Duman, R.S., 2008. VEGF as a potential target for therapeutic intervention in depression. Curr. Opin. Pharmacol. 8, 14–19. Willner, P., 2005. Chronic mild stress (CMS) revisited: consistency and behavioural-neurobiological concordance in the effects of CMS. Neuropsychobiology 52, 90–110.
www.elsevier.com/locate/euroneuro
VEGF regulates antidepressant effects of lamotrigine Rongju Sun a , Nanxin Li b,⁎, Tanshi Li a,⁎⁎ a b
Department of Emergency, General Hospital of PLA, Beijing 100853, China Department of Psychology, Yale University, New Haven, CT 06520, USA
Received 18 July 2011; received in revised form 29 August 2011; accepted 29 September 2011
KEYWORDS Antidepressant; VEGF; CUS; FST; Lamotrigine; NSF
Abstract The anticonvulsant drug lamotrigine has been shown to produce strong antidepressant effects in the treatment of bipolar disorder patients. Our previous studies have demonstrated that brain derived neurotrophic factor (BDNF) signaling plays an important role in regulating its behavioral actions in several rodent models of depression. The current study extends earlier work on BDNF and explores the role of another important neurotrophin vascular endothelial growth factor (VEGF) in regulating the antidepressant actions of lamotrigine. The results showed that chronic administration of 30 mg/kg lamotrigine (14 days) normalized the down-regulated frontal and hippocampal VEGF protein expression as well as the behavioral deficits induced by chronic unpredictable stress. In addition, pharmacological inhibition of VEGF signaling by infusion of SU5416, a selective Flk-1 receptor inhibitor, blocks the antidepressant effects of lamotrigine in all behavioral paradigms. Taken together, this study provides further evidence that VEGF is also an essential regulator for the antidepressant effects of lamotrigine. © 2011 Elsevier B.V. and ECNP. All rights reserved.
1. Introduction Bipolar disorder is a severe neuropsychiatric disease that affects about 2% of the population. Although there are many available mood stabilizers, most of them precipitate the onset of the manic episode and induce rapid cycling of manic and depressive phases (Bourin and Prica, 2007). An ⁎ Correspondence to: N. Li, Department of Psychology, Yale University, New Haven, CT 06520, USA. Tel.: + 1 203 8156200. ⁎⁎ Correspondence to: T. Li, Department of Emergency, General Hospital of PLA, Beijing 100853, China. Tel.: +86 10 66936224. E-mail addresses: [email protected] (N. Li), [email protected] (T. Li).
antiepileptic drug lamotrigine has revealed some antidepressant-like effects in patients with bipolar disorders without precipitating mania (Bowden et al., 1999; Calabrese et al., 1999, 2001). Previous rodent studies with lamotrigine using the forced swim test (FST) (Ali et al., 2003; Bourin et al., 2005; Consoni et al., 2006; Li et al., 2010; Prica et al., 2008) have generated some controversial results, which can largely be explained by the dose responses. Another shortcoming of FST is that it is responsive to acute antidepressant treatments, whereas chronic administration of antidepressants is required for the therapeutic effects (Berton and Nestler, 2006). In contrast to FST, chronic unpredictable stress (CUS) is considered one of the better rodent models of depression, which mimics the
0924-977X/$ - see front matter © 2011 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2011.09.010
VEGF regulates antidepressant effects of lamotrigine anhedonia symptom of depression and is only responsive to chronic antidepressant medications. Using this model, our previous work demonstrated that chronic treatment of lamotrigine (30 mg/kg, 21 days) reversed the anhedonic-like symptoms in the sucrose preference test and anxiogenic-like behaviors in the novelty suppressed feeding test (Li et al., 2011), thus had more convincingly evaluated the behavior actions of lamotrigine in rodent animal models of depression. The neurobiological mechanisms underlying the antidepressant effects of lamotrigine also remain largely obscure. The neurotrophic hypothesis of depression states that stress or depression decreases the expression of certain neurotrophic factors in several cortical and limbic brain regions and antidepressant treatments reverses or even blocks this neurotrophic factor deficit to reverse the neuronal atrophies (Duman, 2004). For example, previous studies by many labs including ours have showed that sub-chronic or chronic treatment of lamotrigine increased brain derived neurotrophic factor (BDNF) expression levels in the frontal cortex and hippocampus (Chang et al., 2009; Li et al., 2010). Although much emphasis have been put on BDNF, recent studies have begun to highlight another neurotrophin vascular endothelial growth factor (VEGF) in the neural mechanism of stress/depression and antidepressant (Greene et al., 2009; Heine et al., 2005; Jin et al., 2002; Storkebaum et al., 2004; Warner-Schmidt and Duman, 2007, 2008). In particular, VEGF is shown to mediate the neurogenic and behavioral actions of common antidepressants including fluoxetine and desipramine (Segi-Nishida et al., 2008; Warner-Schmidt and Duman, 2007). In the current study, we aim to directly test if VEGF signaling is also required for the antidepressant effects of lamotrigine.
2. Experimental procedures 2.1. Animals Male Sprague–Dawley rats (Beijing Laboratory Animal Center) weighing 175–200 g were housed in two and maintained under standard conditions with a 12 h light/dark cycle and ad libitum access to food and water. Two experiments were performed in this study (n = 6 to 8 for each group). The first experiment has two variables: stress condition (CUS and non-stressed, see below) and drug treatment (lamotrigine and vehicle). The second experiment, in which all rats were surgerized and subjected to the CUS procedure, also has two variables: infusion treatment (SU5416 and DMSO) and drug treatment (lamotrigine and vehicle). Behavioral tests were performed from 10 AM to 5 PM concurrent with stated housing conditions. The experiments were carried out in accordance with the guidelines of the Beijing Laboratory Animal Center and the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23).
2.2. Drug treatment Lamotrigine (Sigma, St Louis, MO) was dissolved in a mixture of 0.5% carboxymethylcellulose (Sigma, St Louis, MO), 0.4% Tween 80 (American Bioanalytical, Natick, MA), 0.9% benzylic acid (Sigma, St Louis, MO) and saline, which served as the control vehicle in all behavioral and molecular assays. For CUS/behavior experiments, animals were randomly assigned into different drug treatment groups, receiving vehicle or 30 mg/kg daily administration of lamotrigine. The doses used were based on previous studies (Bourin et al., 2005; Consoni et al., 2006; Li et al., 2010, 2011; Prica, et al.,
425 2008). All drugs were administered intraperitoneally (i.p.) at 1.0 ml/kg. Lamotrigine treatment was initiated one week after onset of CUS experiment and lasted for 14 days. The VEGF-Flk-1 selective antagonist SU5416 (4 mM, Sigma, St Louis, MO) or the vehicle (DMSO, Sigma, St Louis, MO) were delivered in a 1 μl volume at the rate of 0.25 μl i.c.v. with a cannula (26GA, PlasticOne, Roanoke, VA) protruding 0.5 mm beyond the guide cannula (PlasticOne, Roanoke, VA) on days 14, 16, 18, and 20. The dose and time course for SU5416 was based on previous studies (Greene et al., 2009; WarnerSchmidt and Duman, 2007).
2.3. Stererotaxic surgeries Six days before the initiation of the CUS paradigm, rats were anesthetized with Nembutal (i.p. 55 mg/kg, Butler Schein, Dublin, OH) and a single guide cannula was stereotaxically placed into the lateral ventricle (coordinates relative to bregma: −0.9 mm AP, −1.5 mm ML, and −3.3 mm DV from dura) and held in place with aluminum screws and dental cement. Postoperative care consisted in peri-surgerical administration of carprofen (5 mg/kg, Butler Schein, Dublin, OH) and triple antibiotic.
2.4. Chronic unpredictable stress (CUS) procedure In CUS, animals were exposed to a variable sequence of mild and unpredictable stressors (Willner, 2005). Our CUS procedure was successfully used to produce depressive-like behavioral changes (Greene et al., 2009; Warner-Schmidt and Duman, 2007). A total of 10 different stressors are used (two stressors per day for 21 days). The stressors used included rotation on a shaker, placement in a 4 °C ambient, lights off for 3 h (10 AM to 1 PM), lights on overnight, strobe light overnight, aversive odor, 45° tilted cages, food and water deprivation, crowding housing and isolation housing. The same CUS procedure was used for both lamotrigine experiments with or without surgery. Control animals were handled every other day and animal weights were monitored.
2.5. Sucrose preference test (SPT) The SPT consisted of a 48 h exposition (days 20 and 21) of rats to a palatable sucrose solution (1%; Sigma, St. Louis, MO), followed by 4 h of water deprivation on day 22 and a 1 h exposure to two identical bottles, one filled with sucrose solution and the other with water. This procedure was adapted from previous studies and has been used previously in our lab (Warner-Schmidt and Duman, 2007). Sucrose and water consumption were determined by measuring the change in the volume of fluid consumed. Sucrose preference was defined as the ratio of the sucrose versus the total consumption (sucrose and water) during the 1 h exposure.
2.6. Novelty-suppressed feeding test (NSFT) The NSFT was performed as previously described (Greene et al., 2009). After the SPT the rats were food-deprived for 12 h and on day 23 placed in an open field (76.5 cm × 76.5 cm × 40 cm, Plexiglas) with a small amount of food in the center. Animals were allowed to explore the open field for 8 min. The sessions were filmed by a camcorder from above and the latency to feed, specifically, the time it took for the animal to approach and take its first bite of the food, was recorded offline by a stopwatch in seconds. Home cage food intake was also measured as a control.
2.7. VEGF protein detection For the first lamotrigine experiment (without surgery) on day 24 the animals were killed by decapitation, brains were removed and both
426 halves of the frontal cortex and the hippocampal tissue were dissected for protein assays. Tissues were kept on dry ice and stored at − 80 °C. After homogenizing by sonication, VGEF protein level of each sample was quantified by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN) following manufacturer's protocol. The intra- and inter-assay variation coefficients (CV%) of the VEGF ELISA assay were 3.7% and 4.6%, respectively. The average% of recovery of rat VEGF spiked was 99%. VGEF protein level was normalized to the total protein level determined by BCA analysis (Pierce, Rockford, IL). Results were expressed as pictogram of VGEF protein/milligram of total protein.
2.8. Statistical analysis All analyses were performed using SPSS 13.0 software and data were reported as mean ± S.E.M. Analysis of variance (ANOVA) followed by Fisher's post hoc test was conducted to compare group differences. The level of statistical significance was set at p b 0.05.
3. Results 3.1. Chronic lamotrigine treatment restores CUS-induced decrease of VEGF expression Chronic treatment of lamotrigine elevated frontal and hippocampal VEGF protein expression levels in both naïve and CUStreated animals (Fig. 1). The effect of stress was significant in both FC (F(1, 25) = 9.66, p b 0.01) and HC (F(1, 25) = 6.16, p b 0.05), indicating CUS significantly reduced VEGF expression in these two brain regions. The effect of drug was also significant in both FC (F(1, 25) = 11.51, p b 0.01) and HC (F(1, 20) = 11.33, p b 0.01). The interaction between stress and drug was not significant in either FC or HC (FC, p = 0.30; HC, p = 0.75). Further analysis indicated that in CUS-treated rats receiving lamotrigine showed a significant increase of VEGF expression as compared with the CUS group receiving vehicle (FC and HC, both p b 0.05), to a level comparable to non-stressed (NS) control rats (FC, p = 0.84; HC, p = 0.55). Lamotrigine also increased VEGF expression in non-stressed rats (FC and HC, both p b 0.05). The levels of total Flk-1 receptor remained comparable between different treatment groups (see supplemental materials).
R. Sun et al.
3.2. Chronic lamotrigine treatment ameliorates CUS-induced behavioral deficits Consistent with previous findings (Li et al., 2011), chronic treatment of lamotrigine produced robust antidepressant in SPT and NSFT (Fig. 2). For SPT (Fig. 2A), the interaction between stress condition and drug treatment was significant (F(1, 25) = 5.38, p b 0.05). The effect of stress was significant (F(1, 20) = 6.32, p b 0.05), indicating CUS produced potent anhedonia effects. The effect of drug was also significant (F(1, 20) = 7.36, p b 0.05). Further analysis indicated that in CUS-treated rats receiving lamotrigine showed a significant increase in sucrose preference compared with the CUS group receiving vehicle control (p b 0.01), to a level comparable to non-stressed (NS) control rats (p = 0.72). In addition, lamotrigine did not affect sucrose preference in non-stressed control rats (p = 0.76). There was no significant difference in total fluid consumption for the test (data not shown). For NSFT (Fig. 2B), the interaction between stress condition and drug treatment was significant (F(1, 25) = 4.63, p b 0.05). The effect of stress was significant (F(1, 25) = 5.48, p b 0.05), indicating CUS produced anxiogenic effects in rats. The effect of drug was also significant (F(1, 25) = 9.87, p b 0.01). Further analysis indicated that in CUS-treated rats receiving lamotrigine showed a significant decrease in latency to feed as compared with the CUS group receiving vehicle control (pb 0.01), to a level comparable to non-stressed (NS) control rats (p = 0.65). In addition, lamotrigine did not affect latency to feed in non-stressed control rats (p = 0.55). There was no significant difference in home cage food consumption (data not shown).
3.3. Blockade of VEGF-Flk-1 signaling abolishes the antidepressant effects of lamotrigine Blockaded of VEGF-Flk-1 signaling by inhibitor SU5416 completely abolished the antidepressant effects of lamotrigine in SPT and NSFT (Fig. 3). For SPT (Fig. 3A), the interaction between infusion and injection treatment was significant (F(1, 22) = 11.32, p b 0.01). The effect of infusion was significant (F(1, 22) = 5.64, p b 0.05). The effect of injection was also
Figure 1 Chronic lamotrigine treatment increased VEGF protein expression in FC (A) and HC (B). CUS + Lamotrigine rats showed higher FC and HC VEGF expressions to than CUS + Vehicle rats. There was no difference between CUS + Lamotrigine and nonstressed + Vehicle rats. Non-stressed + Lamotrigine rats also showed increased VEGF level than non-stressed + Vehicle rats. Results were expressed as mean + S.E.M., *, p b 0.05; #, non-significant, as compared with vehicle treated controls. n = 6 to 8 for each group. NS denotes non-stressed, CUS denotes chronic unpredictable stress, Veh denotes vehicle treatment, and Lam denotes lamotrigine treatment.
VEGF regulates antidepressant effects of lamotrigine
427
Figure 2 Chronic lamotrigine treatment produced robust antidepressant effects in SPT (A) and NSFT (B). CUS + Lamotrigine rats displayed higher sucrose preference and reduced latency to feed than CUS + Vehicle rats. There was no difference of sucrose preference and latency to feed between CUS + Lamotrigine and non-stressed + Vehicle rats. Results were expressed as mean + S.E.M., **, p b 0.01; #, non-significant, as compared with vehicle treated controls. n = 6 to 8 for each group. NS denotes non-stressed, CUS denotes chronic unpredictable stress, Veh denotes vehicle treatment, and Lam denotes lamotrigine treatment.
significant (F(1, 20) = 9.69, p b 0.01). Further analysis indicated that consistent with the last experiment, CUS-treated rats receiving lamotrigine showed a significant increase in sucrose preference compared with the CUS group receiving vehicle control (p b 0.01). This antidepressant effect of lamotrigine was completely blocked by infusion of the VEGF-Flk-1 antagonist: the CUS + lamotrigine + SU5416 group was not significantly different from the CUS + vehicle + DMSO or CUS + vehicle + SU5416 groups (p = 0.62, p = 0.86). There was no significant difference in total fluid consumption for the 1 h test (data not shown). For NSFT (Fig. 3B), the interaction between infusion and injection treatment was significant (F(1, 22) = 5.53, p b 0.05). The effect of infusion was significant (F(1, 22) = 6.77, p b 0.05). The effect of injection was also significant (F(1, 22) = 5.53, p b 0.05). Further analysis indicated that similar to last experiment, CUS-treated rats receiving lamotrigine showed a significant decrease of latency to feed as compared with the CUS group receiving vehicle control (p b 0.01). This antidepressant effect of lamotrigine was completely blocked by infusion of the VEGF-Flk-1 antagonist: the CUS + lamotrigine + SU5416 group was not significantly different from the CUS + vehicle + DMSO or CUS + vehicle + SU5416 groups (p = 0.76, p = 0.70). There was no
significant difference in home cage food consumption (data not shown).
4. Discussion The current study corroborates our previous studies and demonstrates that the mood stabilizer lamotrigine produces antidepressant effects in chronic unpredictable stress model of depression. Chronic lamotrigine treatment also up-regulates frontal and hippocampal VEGF expression levels in both nonstressed and stressed animals and completely reversed stressinduced VEGF deficits. Furthermore, pharmacological inhibition of VEGF-Flk-1 signaling completely abolishes antidepressant actions of lamotrigine. The dose of lamotrigine largely determines its therapeutic effects. In consistent with our recent studies (Li et al., 2010, 2011), we have used a relatively high dose (30 mg/kg) dose for the chronic administration. Previous studies reveal that lamotrigine can only decrease immobility in FST at high (10 mg/kg and above) but not lower doses (1 to 5 mg/kg) (Ali et al., 2003; Bourin et al., 2005; Li et al., 2010;
Figure 3 Blockade of VEGF-Flk-1 signaling abolished antidepressant actions of lamotrigine in SPT (A) and NSFT (B). CUS + DMSO + Lamotrigine rats displayed higher sucrose preference and reduced latency to feed than CUS + DMSO + Vehicle rats. There was no difference of su crose preference and latency to feed and immobility between CUS+ SU5416 + Lamotrigine and CUS+ SU5416 + Vehicle as well as CUS + DMSO + Vehicle rats. Results were expressed as mean + S.E.M., **, p b 0.01; #, non-significant, as compared with vehicle treated controls. n = 6 to 8 for each group. NS denotes non-stressed, CUS denotes chronic unpredictable stress, Veh denotes vehicle treatment, and Lam denotes lamotrigine treatment.
428 Prica et al., 2008). The 30 mg/kg dose used in the present study is well tolerated and does not cause any locomotor abnormalities (Li et al., 2010). The two rodent models currently used are only responsive to chronic antidepressant and thus better reflect the clinical therapeutic responses than behavioral despair models including FST. CUS is widely considered a better stress model since rodents develop a range of fundamental behavioral and neurochemical changes that resemble core symptoms of depression (Willner, 2005). One of the most evident abnormalities is reduced motivation for incentive stimuli, clinically known as anhedonia. Novelty suppressed feeding test is a model of anxiety and is commonly used in depression studies due to the high co-morbidity of these two diseases (Schmidt and Duman, 2007). Consistent with our previous report (Li et al., 2011), CUS produces severe anhedonic- and anxiogeniclike in rats, both of which are completely ameliorated by chronic lamotrigine treatment. Lamotrigine neither alters the behavior in non-stressed animals nor affects total fluid consumption or home-cage food intake, indicating the effects observed in the SPT and NSFT are specific to its antidepressant actions rather than its effects on metabolism. Therefore, together with recent findings by our and other laboratories, the current findings provide additional evidence on the preclinical antidepressant effects of lamotrigine. Most of previous studies on the neurobiological mechanism of lamotrigine have focused on BDNF and consistently demonstrated that chronic treatment of lamotrigine markedly increases BDNF mRNA and protein expression in several cortical and limbic regions including the frontal cortex and hippocampus (Li et al., 2011; Rao and Rapoport, 2009). The current study, for the first time, shows that lamotrigine can also activate another important neutrophic factor VEGF in the brain. Originally discovered as an endothelial survival marker, VEGF has recently shown strong neurogenic and neuroprotective effects and modulates synaptic activities and many hippocampal-dependent functions (Jin et al., 2002; McCloskey et al., 2005; Newton et al., 2003; Schanzer et al., 2004; Storkebaum et al., 2004). VGEF binds to two tyrosine receptors Flk-1 and Flt-1, of which the former is mainly expressed in the brain, in particularly the endothelial cells and neural progenitor cells in the HC (Warner-Schmidt and Duman, 2008). In particular, the up-regulation of VEGF-Flk-1 signaling is required for the behavioral and neurogenic effects of common antidepressants (Greene et al., 2009; WarnerSchmidt and Duman, 2007). The present study demonstrates that similar to fluoxetine and electroconvulsive shock (Warner-Schmidt and Duman, 2007), and chronic lamotrigine treatment can increase VEGF protein expression in both FC and HC, two important regions in the neural circuitry of depression. VEGF gene contains CRE and its expression can be stimulated by the activation of CREB (Greene et al., 2009). Previous studies have demonstrated that a variety of mood stabilizers, including lithium and valproate acid, can robustly increase phospho-CREB expression in the brain (Einat et al., 2003). It is reasonable to predict that lamotrigine may also regulate the expression of VEGF via activation of pCREB, though future studies were needed to confirm this hypothesis. Additional studies using double-label florescent in situ hybridization (FISH, with markers such as NeuN and GFAP) are needed to localize the source of VEGF release (neuron or glia) triggered by lamotrigine and to examine whether VEGF mRNA levels were
R. Sun et al. regulated or not. Furthermore, a recent study showed that veratrine, a sodium channel opener, can block the antidepressant effects of lamotrigine in FST (Prica et al., 2008). It will be interesting to study if there is any neurobiological connection among sodium channel, pCREB, and VEGF underlying the therapeutic effects of lamotrigine. More importantly, lamotrigine completely reverses the stressed-induced VEGF deficits to comparable levels of non-stressed controls. The neurotrophic hypothesis of depression postulates that stress/depression down-regulates the neurotrophic functions, which lead to structure and functional deficiencies in several key regions including FC and HC. In contrast, antidepressant medications produce therapeutic effects via reversing aforementioned neurotrophic changes and exerting neuroprotective effects (Duman, 2004). Chronic stress can decrease FC and HC VEGF levels, which can be alleviated by chronic antidepressant treatment (Greene et al., 2009; Heine et al., 2005; Warner-Schmidt and Duman, 2007). A recent study showed that chronic continuous exposure to corticosterone (CORT, a natural glucocorticoid) significantly reduced Flk-11 protein levels both in cultured cortical neurons and mouse frontal cortex (Howell et al., 2011). In the current study, we did not find any significant alteration of total Flk-1 proteins in frontal cortex or hippocampus. We reason that CUS and CORT are two different kinds of stress and it is possible that instead of regulating the total expression levels, CUS may regulate the activity of this receptor. Unfortunately all phosphor-Flk-1 antibodies we have attempted to use failed to generate consistent and reliable results and thus it remained an open question to be explored. Previous studies indicate that increasing VEGF signaling may a common pathway underlying the neurobiological mechanisms of various antidepressants, including lamotrigine. To directly test this hypothesis, we pharmacologically block the VEGF signaling in the brain by infusing Flk-1 receptor antagonist SU5416 and then investigate whether the infusion affects the behavioral effects of lamotrigine. VEGF preferentially binds to Flk-1 than Flt-1 receptor in the brain. Blockade of VEGF-Flk-1 signaling by intracerebroventricular infusion of the selective inhibitor SU5416 abolishes the antidepressant effects of fluoxetine and desipramine in several rodent models of depression, including CUS/SPT, NSFT, and FST (Greene et al., 2009; Warner-Schmidt and Duman, 2007). In the current study we find that SU5416 can also block the behavioral effects of lamotrigine in SPT and NSFT, indicating that VEGF-Flk-1 signaling indeed mediates the antidepressant actions of lamotrigine. Using a pharmacological inhibition approach, we have previously demonstrated that BDNF-TrkB signaling is also required for the therapeutic effects of lamotrigine in rodent models of depression (Li et al., 2011). It raises an interesting question on whether BDNF or VEGF (or both) are important for the antidepressant actions of lamotrigine. Previous studies have demonstrated that similar to lamotrigine, the therapeutic effects of many antidepressants, including the selective serotonin reuptake inhibitor fluoxetine, can activate both BDNF and VEGF signaling pathways in the brain to produce antidepressant effects (Greene et al., 2009; Pilar-Cuellar et al., 2011). Together with the current findings, we postulate that both pathways are necessary for the antidepressant actions of lamotrigine and neither pathway by itself is sufficient to
VEGF regulates antidepressant effects of lamotrigine mediate such actions. Future studies utilizing a combination of pharmacological inhibition and conditional knockout mice (since constitutive homozygous BDNF and VEGF knockout are both lethal) are needed to further address this issue. In addition, the specificity and potential cross-talk between different neurotrophic factor receptor antagonists warrant further investigation. For example, the effects of SU5416 on BDNF and TrkB and the effects of K252a on VEGF and Flk-1 should be extensively studied to help support the wide application of these inhibitors. There are a few limitations in this study. With the current drug delivery approach, it is difficult to determine in which part of brain is essentially involved in this signaling pathway. The current results suggest that FC and HC are good candidates for future studies with local microinjection to determine the regional specificity. In addition, although SU5416 is generally considered a reliably selective inhibitor of Flk1 receptor, like another commonly used inhibitor SU1498, the specificity of pharmacological inhibitors is still far from optimal. Other approaches, including genetic-mutant mice, should be used to more rigorously address the role of VEGF signaling in regulating antidepressant effects of lamotrigine. Taken together, the present study demonstrates that chronic treatment of lamotrigine increases FC and HC VEGF expressions and ameliorates CUS-induced behavioral deficits. Blockade of VEGF-Flk-1 signaling abolishes its therapeutic effects. The current findings expand our understanding of the potential neurobiological mechanisms of antidepressant actions of lamotrigine.
Role of the funding source Funding for this study was provided by National Natural Science Foundation of China and American Psychological Association; none of these grant sources had any further role in study design, in the collection, analysis and interpretation of data, in the writing of the report and in the decision to submit the paper for publication.
Contributors Authors TL and NL designed the study and wrote the protocol. Author RS and NL managed the literature searches and analyses. Author RS and NL undertook the statistical analysis, and authors TL and NL wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.
Conflict of interest All authors of this manuscript declare no conflict of interest.
Acknowledgments The current study was funded by grants from National Natural Science Foundation of China (30700869, 30840039) and American Psychological Association.
Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.euroneuro.2011.09.010.
429
References Ali, A., Pillai, K.K., Pal, S.N., 2003. Effects of folic acid and lamotrigine therapy in some rodent models of epilepsy and behaviour. J. Pharm. Pharmacol. 55, 387–391. Berton, O., Nestler, E.J., 2006. New approaches to antidepressant drug discovery: beyond monoamines. Nat. Rev. Neurosci. 7, 137–151. Bourin, M., Prica, C., 2007. The role of mood stabilisers in the treatment of the depressive facet of bipolar disorders. Neurosci. Biobehav. Rev. 31, 963–975. Bourin, M., Masse, F., Hascoet, M., 2005. Evidence for the activity of lamotrigine at 5-HT(1A) receptors in the mouse forced swimming test. J. Psychiatry Neurosci. 30, 275–282. Bowden, C.L., Mitchell, P., Suppes, T., 1999. Lamotrigine in the treatment of bipolar depression. Eur. Neuropsychopharmacol. 9 (Suppl. 4), S113–S117. Calabrese, J.R., Bowden, C.L., Sachs, G.S., Ascher, J.A., Monaghan, E., Rudd, G.D., 1999. A double-blind placebo-controlled study of lamotrigine monotherapy in outpatients with bipolar I depression. Lamictal 602 study group. J. Clin. Psychiatry 60, 79–88. Calabrese, J.R., Shelton, M.D., Rapport, D.J., Kujawa, M., Kimmel, S.E., Caban, S., 2001. Current research on rapid cycling bipolar disorder and its treatment. J. Affect. Disord. 67, 241–255. Chang, Y.C., Rapoport, S.I., Rao, J.S., 2009. Chronic administration of mood stabilizers upregulates BDNF and bcl-2 expression levels in rat frontal cortex. Neurochem. Res. 34, 536–541. Consoni, F.T., Vital, M.A., Andreatini, R., 2006. Dual monoamine modulation for the antidepressant-like effect of lamotrigine in the modified forced swimming test. Eur. Neuropsychopharmacol. 16, 451–458. Duman, R.S., 2004. Role of neurotrophic factors in the etiology and treatment of mood disorders. Neuromolecular Med. 5, 11–25. Einat, H., Manji, H.K., Gould, T.D., Du, J., Chen, G., 2003. Possible involvement of the ERK signaling cascade in bipolar disorder: behavioral leads from the study of mutant mice. Drug News Perspect. 16, 453–463. Greene, J., Banasr, M., Lee, B., Warner-Schmidt, J., Duman, R.S., 2009. Vascular endothelial growth factor signaling is required for the behavioral actions of antidepressant treatment: pharmacological and cellular characterization. Neuropsychopharmacology 34, 2459–2468. Heine, V.M., Zareno, J., Maslam, S., Joels, M., Lucassen, P.J., 2005. Chronic stress in the adult dentate gyrus reduces cell proliferation near the vasculature and VEGF and Flk-1 protein expression. Eur. J. Neurosci. 21, 1304–1314. Howell, K.R., Kutiyanawalla, A., Pillai, A., 2011. Long-term continuous corticosterone treatment decreases VEGF receptor-2 expression in frontal cortex. PLoS One 6, e20198. Jin, K., Zhu, Y., Sun, Y., Mao, X.O., Xie, L., Greenberg, D.A., 2002. Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc. Natl. Acad. Sci. U. S. A. 99, 11946–11950. Li, N., He, X., Qi, X., Zhang, Y., He, S., 2010. The mood stabilizer lamotrigine produces antidepressant behavioral effects in rats: role of brain-derived neurotrophic factor. J. Psychopharmacol. 23, 1772–1778. Li, N., He, X., Zhang, Y., Qi, X., Li, H., Zhu, X., He, S., 2011. Brainderived neurotrophic factor signalling mediates antidepressant effects of lamotrigine. Int. J. Neuropsychopharmacol. 1–8. McCloskey, D.P., Croll, S.D., Scharfman, H.E., 2005. Depression of synaptic transmission by vascular endothelial growth factor in adult rat hippocampus and evidence for increased efficacy after chronic seizures. J. Neurosci. 25, 8889–8897. Newton, S.S., Collier, E.F., Hunsberger, J., Adams, D., Terwilliger, R., Selvanayagam, E., Duman, R.S., 2003. Gene profile of electroconvulsive seizures: induction of neurotrophic and angiogenic factors. J. Neurosci. 23, 10841–10851.
430 Pilar-Cuellar, F., Vidal, R., Pazos, A., 2011. Subchronic treatment with fluoxetine and the 5-HT(2A) antagonist ketanserin upregulates hippocampal BDNF and beta-catenin in parallel with antidepressant-like effect. Br. J. Pharmacol. doi:10.1111/j.14765381.2011.01516.x (Electronic publication ahead of print). Prica, C., Hascoet, M., Bourin, M., 2008. Antidepressant-like effect of lamotrigine is reversed by veratrine: a possible role of sodium channels in bipolar depression. Behav. Brain Res. 191, 49–54. Rao, J.S., Rapoport, S.I., 2009. Mood-stabilizers target the brain arachidonic acid cascade. Curr. Mol. Pharmacol. 2, 207–214. Schanzer, A., Wachs, F.P., Wilhelm, D., Acker, T., Cooper-Kuhn, C., Beck, H., Winkler, J., Aigner, L., Plate, K.H., Kuhn, H.G., 2004. Direct stimulation of adult neural stem cells in vitro and neurogenesis in vivo by vascular endothelial growth factor. Brain Pathol. 14, 237–248. Schmidt, H.D., Duman, R.S., 2007. The role of neurotrophic factors in adult hippocampal neurogenesis, antidepressant treatments
R. Sun et al. and animal models of depressive-like behavior. Behav. Pharmacol. 18, 391–418. Segi-Nishida, E., Warner-Schmidt, J.L., Duman, R.S., 2008. Electroconvulsive seizure and VEGF increase the proliferation of neural stem-like cells in rat hippocampus. Proc. Natl. Acad. Sci. U. S. A. 105, 11352–11357. Storkebaum, E., Lambrechts, D., Carmeliet, P., 2004. VEGF: once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays 26, 943–954. Warner-Schmidt, J.L., Duman, R.S., 2007. VEGF is an essential mediator of the neurogenic and behavioral actions of antidepressants. Proc. Natl. Acad. Sci. U. S. A. 104, 4647–4652. Warner-Schmidt, J.L., Duman, R.S., 2008. VEGF as a potential target for therapeutic intervention in depression. Curr. Opin. Pharmacol. 8, 14–19. Willner, P., 2005. Chronic mild stress (CMS) revisited: consistency and behavioural-neurobiological concordance in the effects of CMS. Neuropsychobiology 52, 90–110.