Valproate Inhibits Methamphetamine Induced Hyperactivity via Glycogen Synthase Kinase 3β Signaling in the Nucleus Accumbens Core

<div><p>Valproate (VPA) has recently been shown to influence the behavioral effects of psycho-stimulants. Although glycogen synthase kinase 3β (GSK3β) signaling in the nucleus accumbens (NAc) plays a key role in mediating dopamine (DA)-dependent behaviors, there is less direct evidence that how VPA acts on the GSK3β signaling in the functionally distinct sub-regions of the NAc, the NAc core (NAcC) and the NAc shell (NAcSh), during psycho-stimulant-induced hyperactivity. In the present study, we applied locomotion test after acute methamphetamine (MA) (2 mg/kg) injection to identify the locomotor activity of rats received repeated VPA (300 mg/kg) pretreatment. We next measured phosphor-GSK3β at serine 9 and total GSK3β levels in NAcC and NAcSh respectively to determine the relationship between the effect of VPA on MA-induced hyperlocomotor and changes in GSK3β activity. We further investigated whether microinjection of VPA (300 μg/0.5 μl/side, once daily for 7 consecutive days) into NAcC or NAcSh could affect hyperactivity induced by MA. Our data indicated that repeated VPA treatment attenuated MA-induced hyperlocomotor, and the effect was associated with decreased levels of phosphorylated GSK3β at Ser 9 in the NAcC. Moreover, repeated bilateral intra-NAcC, but not intra-NAcSh VPA treatment, significantly attenuated MA-induced hyperactivity. Our results suggested that GSK3β activity in NAcC contributes to the inhibitory effects of VPA on MA-induced hyperactivity.</p></div

Microinjection of Valproic Acid into the Ventrolateral Orbital Cortex Enhances Stress-Related Memory Formation

<div><p>There is collecting evidence suggesting that the process of chromatin remodeling such as changes in histone acetylation contribute to the formation of stress-related memory. Recently, the ventrolateral orbital cortex (VLO), a major subdivision of orbitofrontal cortex (OFC), was shown to be involved in antidepressant-like actions through epigenetic mechanisms. Here, we further investigated the effects of the histone deacetylase inhibitor (HDACi) valproic acid (VPA) on stress-related memory formation and the underlying molecular mechanisms by using the traditional two-day forced swimming test (FST). The results showed that VPA significantly increased the immobility time on day 2 when infused into the VLO before the initial forced swim stress on day 1. The learned immobility response to the stress was associated with increased phosphorylation of extracellular signal-regulated kinase (ERK) in VLO and hippocampus on the first day. The levels of phosphorylated ERK (phospho-ERK) in VLO and hippocampus were significantly decreased when retested 24 h later. The pretreatment with intra-VLO VPA infusion further reduced the activation of ERK on day 2 and day 7 compared with the saline controls. Moreover, the VPA infusion pretreatment also induced a significantly decreased BDNF level in the VLO on day 2, whereas no change was detected in the hippocampus. These findings suggest that VPA enhance the memories of emotionally stressful events and the ERK activity is implicated in stimulating adaptive and mnemonic processes in case the event would recur.</p> </div

Effects of repeated microinjections of VPA into NAcC and NAcSh on MA-induced hyper-locomotor activity.

<p>Repeated pre-treatment of VPA (300μg/0.5μl/side, consecutive seven days, intracranial injection once a day) into NAcC (A) but not NAcSh (B) significantly attenuates the effect of MA injection (2 mg/kg, intraperitoneal) on locomotor activity compared with saline (SAL) controls. MA induced hyper-locomoter in rats with SAL infusion in both NAcC and NAcSh infusions (SAL_NAcC-MA vs. SAL_NAcC-SAL, <i>p</i> < 0.05; SAL_NAcSh-MA vs. VPA_NAcSh-SAL, <i>p</i> < 0.05 Bonferroni post hoc test). VPA did not affect locomotor activity without MA administration (SAL_NAcC-SAL vs. SAL_NAcC-SAL, <i>p</i> > 0.05; SAL_NAcSh-SAL vs. VPA_NAcSh-SAL, <i>p</i> > 0.05 Bonferroni post hoc test). VPA infusions in NAcC but not NAcSh caused inhibitory effect on MA induced hyper-locomotion compared with saline controls (SAL_NAcC-MA vs. VPA_NAcC-MA, <i>p</i> < 0.05; SAL_NAcSh-MA vs. VPA_NAcSh-MA, <i>p</i> > 0.05 Bonferroni post hoc test). However, there was no significant difference between rats given VPA infusions in NAcSh and saline controls (SAL_NAcSh-MA vs. VPA_NAcSh-MA, <i>p</i> > 0.05, Bonferroni post hoc test). Data are expressed as mean ± SEM, n = 8 per group, *** <i>p</i> < 0.001 versus SAL-SAL, ^#<i>p</i> < 0.05 versus SAL-MA, one-way ANOVA followed by Bonferroni's Multiple Comparison Test.</p

The influence of intro-VLO injection of VPA on forced swim stress induced stress-related memory.

<p>A: Time line of the experimental procedure. The syringe indicated that the VPA (300 µg) or saline were bilateral intro-VLO injected 30 min before the initial forced swim stress. The black arrows indicate the exposure of forced swim stress. B: The immobility time at the different test day (day 1, 2 or 7). The immobility time of VPA (300 µg) treated rats were compared with the saline group at the same time point. All data are expressed as the mean ± SEM. **<i>p</i><0.01 and ***<i>p</i><0.001 versus pre-test or saline group.</p

Schematic frame of the drug treatment and behavioral procedure.

<p>The black arrows indicate the seven consecutive days VPA pre-treatment (either 300 mg/kg, intraperitoneal injection or 300μg/0.5μl/side, intracranial injection) or saline controls before the MA injection and locomotor test.</p

Effect of pre-treatment with VPA on MA-induced activation of GSK3β signaling in NAcC and NAcSh.

<p>Representative blots and summary histograms of phospho-GSK3β at Ser 9 (pGSK3b) and total GSK3β (GSK3b) from NAcC (A) and NAcSh (B) tissues from repeated saline (SAL) and VPA pre-treatment rats which then received SAL or MA injection. SAL-SAL group levels were set at 1 for quantifications. Data were expressed as mean ± SEM, n = 8 per group, ** <i>p</i> < 0.01 versus SAL-SAL, ^#<i>p</i> < 0.05 versus SAL-MA, one-way ANOVA followed by Bonferroni's Multiple Comparison Test.</p

Effect of pre-treatment with VPA on MA-induced hyper-locomotor activity.

<p>Repeated pre-treatment of VPA (300 mg/kg, seven consecutive days, intraperitoneal once a day) significantly attenuates the effect of MA injection (2 mg/kg, intraperitoneal) on locomotor activity compared with saline (SAL) controls. Data are expressed as mean ± SEM, n = 8 per group, ** <i>p</i> < 0.01 versus SAL-SAL, one-way ANOVA followed by Bonferroni's Multiple Comparison Test.</p

Effect of intra-VLO VPA injection on protein levels of ERK and BDNF in hippocampus.

<p>The phosphorylation state of ERK was expressed as phospho-ERK to total-ERK. The BDNF results are normalized to β-actin. Sham group was set as 100%. The data are expressed as the mean ± SEM. ***<i>p</i><0.0001, difference between sham and saline group on day 1; ^#<i>p</i><0.05, ^###<i>p</i><0.0001, difference from day 1 within the same groups. **<i>p</i><0.01, ***<i>p</i><0.0001, difference between VPA and stress groups.</p

Effect of intra-VLO VPA injection on protein levels of ERK and BDNF in VLO assessed by western blotting.

<p>The phosphorylation state of ERK was expressed as phospho-ERK to total-ERK. The BDNF results are normalized to β-actin. Sham group was set as 100%. The data are expressed as the mean ± SEM. ***<i>p</i><0.0001, difference between sham and saline group on day 1; ^###<i>p</i><0.0001, difference from day 1 within the same groups. *<i>p</i><0.05, ***<i>p</i><0.0001, difference between VPA and stress groups.</p