Dose-Dependent Neuroprotective Effects of Valproate on Motor Function and Striatal D2 Receptor Stability in a 6-OHDA Rat Model of Parkinson’s Disease

Abstract

Parkinson’s disease (PD) is characterized by progressive degeneration of nigrostriatal dopaminergic neurons, leading to motor dysfunction and compensatory postsynaptic dopamine receptor alterations. Valproic acid (VPA), a histone deacetylase inhibitor, has shown neuroprotective properties; however, its dose-dependent effects on dopaminergic integrity and dopamine D2 receptor (D2R) regulation remain unclear. Adult male Wistar rats received VPA (200 or 400 mg/kg, p.o.) or vehicle for 20 days prior to unilateral 6-hydroxydopamine (6-OHDA) lesioning. Motor performance was evaluated using the beam balance test, exploratory behavior in the open field, striatal dopamine levels by PLC-electrochemical detection, and D2R protein expression by Western blot. The 6-OHDA lesion induced marked motor deficits, reduced striatal dopamine content, and significantly increased D2R expression. VPA at 200 mg/kg produced only minor, non-significant effects. In contrast, VPA at 400 mg/kg preserved motor performance, attenuated dopamine depletion, and normalized striatal D2R expression. These findings demonstrate a clear dose-dependent neuroprotective effect of VPA and indicate that stabilization of postsynaptic D2R expression accompanies preservation of dopaminergic terminals in the 6-OHDA rat model.

1. Introduction

Parkinson’s disease (PD) is the second-most prevalent chronic neurodegenerative disorder worldwide, impacting millions of people, and its incidence may double by 2040 [1,2]. The main issue is the gradual loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), which leads to a significant dopamine (DA) deficiency in the striatum. This deficiency causes the typical motor symptoms: bradykinesia, rigidity, and resting tremor [3]. Current treatments, mainly L-DOPA therapy, help reduce symptoms but do not delay disease progression. Prolonged use of L-DOPA can result in serious motor side effects such as dyskinesias and motor fluctuations [4].

Valproic acid (VPA) is a common medication for epilepsy and bipolar disorder and is now being considered for new uses [5]. VPA primarily acts by enhancing GABAergic neurotransmission and, importantly, by strongly inhibiting histone deacetylases (HDACs) [6]. Blocking HDACs increases histone acetylation, opens chromatin, and boosts expression of key survival genes. VPA increases levels of neurotrophic factors, such as Brain-Derived Neurotrophic Factor (BDNF), and anti-apoptotic proteins, such as Bcl-2, thereby providing a robust means of protecting neurons in PD models [7].

Despite its promise, important questions about VPA remain. First, the optimal dose for VPA’s neuroprotective effect in PD remains debated [8]. Low doses may not induce sufficient epigenetic changes, whereas high doses can be toxic. Additionally, although preserving DA neurons is a common goal, the effect of VPA on postsynaptic receptor stability is often overlooked. In the 6-OHDA model, DA loss leads to an increase in the density of dopamine D2 receptors (D2Rs) on striatal neurons, a phenomenon known as denervation supersensitivity [9,10]. This change is a key marker of PD progression and is associated with motor dysfunction [8,11]. Preventing D2R upregulation could be a better marker of true neuroprotection.

This study was designed to rigorously investigate the dose-dependent effects of VPA (200 versus 400 mg/kg) on motor behavior and dopaminergic integrity in the 6-OHDA rat model. Importantly, novel evidence is presented by simultaneously analyzing how these distinct doses differentially modulate D2R protein expression in the striatum, thereby linking the presynaptic neuroprotective action of VPA to a functional postsynaptic outcome.

2. Results

2.1. VPA 400 mg/kg Robustly Prevents Motor Deficits

In the balance beam test, the 6-OHDA group exhibited severe, persistent motor impairment (mean score > 3 on a six-point scale). The VPA-200 group demonstrated only minimal and non-significant improvement (* p < 0.002 vs. SHAM). Notably, the VPA-400 group showed no significant deficits compared to SHAM (p < 0.07), indicating effective preservation of fine motor coordination (Figure 1).

2.2. Normalization of Exploratory Behavior

The 6-OHDA group exhibited an anxiety-like phenotype in the open field test, as indicated by a significant reduction in time spent in the center zone and an increase in time spent in the periphery and corners. VPA-200 did not fully restore normal exploratory patterns. In contrast, VPA-400 maintained a normal zonal distribution of activity, comparable to SHAM (Figure 2).

2.3. Neuroprotection of Dopaminergic Terminals

HPLC-EC analysis confirmed the effectiveness of the lesion model, revealing a significant loss of striatal dopamine in both the 6-OHDA and VPA-200 groups (** p < 0.001 vs. SHAM). VPA-400 treatment provided substantial neuroprotection, preserving dopamine levels significantly more than the other lesion groups in both the striatum (Figure 3A) and the motor cortex (Figure 3B), suggesting preservation of the structural integrity of the nigrostriatal projection.

2.4. Modulation of Striatal D2 Receptor Expression

To determine the postsynaptic consequence of VPA treatment, we analyzed D2R protein levels in the striatum. 6-OHDA Group: Exhibited a significant upregulation of D2R expression (approximately 180% of SHAM control), a definitive molecular signature of “denervation supersensitivity” due to chronic DA deprivation. VPA-200 Group: Showed a partial, yet still significantly elevated, level of D2R expression compared to SHAM. VPA-400 Group: Displayed D2R protein expression levels that were remarkably similar to the SHAM group.

This normalization of D2R expression demonstrates that preservation of presynaptic dopamine release by high-dose VPA effectively prevented maladaptive postsynaptic receptor upregulation (Figure 4).

3. Discussion

This study shows that the neuroprotective effects of valproic acid in the 6-OHDA PD model depend on the dose. The lower dose (200 mg/kg) yielded only slight, non-significant benefits, whereas the higher dose (400 mg/kg) provided strong protection across behavioral, chemical, and molecular tests. This clear dose-response pattern is consistent with VPA’s known action as an HDAC inhibitor. On the other hand, VPA does not appear to reproduce the motor phenotype of Parkinson’s disease in healthy animals, and in fact, in pathological models, it tends to protect and improve motor behaviors; the only evidence of a negative motor effect in healthy rats is the potentiation of catalepsy when co-administered with a D2 antagonist [8,12,13].

3.1. The Critical Role of Dose in Epigenetic Modulation

The lack of effect at 200 mg/kg is likely due to the drug’s metabolism. VPA must reach sufficiently high concentrations in the blood and brain to cross the blood–brain barrier and inhibit HDACs.

The finding in the present study, that 400 mg/kg is required to achieve neuroprotective effects, is consistent with those obtained by Ximenes et al. [8], who observed a similar threshold for behavioral improvement. The 400 mg/kg dose is effective because it induces the required epigenetic changes. It increases histone H3 acetylation in the SNpc and striatum, thereby activating key survival genes such as BDNF and Gdnf [13,14]. This boost supports dopaminergic neurons and helps them survive 6-OHDA-induced damage [8,15].

The efficacy of the 400 mg/kg dose suggests the existence of a pharmacological threshold necessary for VPA to reach sufficient brain concentrations in order to effectively inhibit HDACs. Previous studies indicate that VPA-induced hyperacetylation of histone H3 promotes the transcription of essential neurotrophic factors, such as BDNF and GDNF, which are critical for the survival of dopaminergic neurons under oxidative stress. Our results support the hypothesis of “epigenetic priming,” where the 3-week pretreatment established a neuroprotective environment prior to neurotoxin application [7,12].

3.2. D2 Receptor Stabilization as a Marker of Dopaminergic Functional Integrity Preservation

A key new finding in this study is that VPA at 400 mg/kg restores dopamine D2 receptor (D2R) levels in the striatum to normal levels. In PD, dopamine loss typically leads to increased D2R expression on medium spiny neurons (supersensitivity). Maintaining D2R at normal levels in the VPA-400 group indicates that dopamine signaling is preserved. By protecting presynaptic dopamine release, the cause of postsynaptic D2R increase is removed. This is important because D2R supersensitivity is associated with L-DOPA-induced dyskinesia [16]. These results suggest that VPA-400 provides more comprehensive neuroprotection by preserving the integrity of the entire nigrostriatal pathway [17].

From the above, the most significant contribution of the present study is the normalization of D2 receptor expression. In the 6-OHDA group, we observed significant increase in D2R, a classic marker of denervation hypersensitivity. This upward regulation occurs mainly in the medium spiny neurons (MSN) of the striatum as a compensatory response to the loss of dopaminergic input.

In a healthy system, D2Rs also exist as presynaptic auto receptors that provide inhibitory feedback. The total level of D2R protein measured by Western blot in the 6-OHDA group represents the net sum of (a) loss of presynaptic auto receptors due to synaptic terminal death and (b) massive increase in postsynaptic D2 receptors as compensatory upregulation.

The normalization of these levels in the VPA-400 mg/kg group indicates dual success: it probably preserved the presynaptic terminal and its auto receptors, simultaneously, eliminating the DA deprivation signal that triggers postsynaptic hypersensitivity. This stabilization is clinically relevant, as D2R hypersensitivity is a key factor in the development of L-DOPA-induced dyskinesias (LIDs) [13,18].

A critical finding was the significant preservation of DA levels in the striatum and motor cortex in the VPA-400 group. However, the absence of immunohistochemistry for tyrosine hydroxylase in this study limits the direct quantification of neuronal bodies in the SNpc. Nevertheless, HPLC-EC data serve as a highly sensitive marker of the functional integrity of nigrostriatal terminals. The recovery of DA levels suggests that VPA prevented the physical degeneration of axons or maintained their metabolic and biosynthetic capacity [18,19].

3.3. Mechanisms of Action: A Dual Neuroprotective Strategy

VPA’s neuroprotection in this model is probably mediated by a dual, synergistic mechanism: (I) Epigenetic Modulation: Inhibiting HDACs not only promotes neurotrophic factor expression but also modulates the neuroinflammatory response, notably by suppressing pro-inflammatory cytokines (TNF-alpha) and activating anti-apoptotic pathways (Bcl-2, Akt signaling) [20,21]. (II) GABAergic Enhancement: While not the primary focus, VPA’s general action to enhance GABAergic transmission may help reduce excitotoxicity in the basal ganglia circuit, thereby lowering the overall vulnerability of the dopaminergic system to 6-OHDA [22].

The phenomenon of denervation hypersensitivity observed in the 6-OHDA group, characterized by a massive increase in D2 receptor density (~180%), is a maladaptive homeostatic response to chronic dopamine deficiency in the synaptic cleft. This upregulation of postsynaptic receptors in medium spiny neurons may be the pathophysiological substrate of motor fluctuations and dyskinesias.

Our results demonstrate that treatment with VPA 400 mg/kg not only preserves striatal dopamine levels but also maintains the stability of the postsynaptic architecture. By stabilizing D2R expression at levels similar to those in the SHAM group, VPA prevents the development of this state of hypersensitivity. This suggests that valproic acid acts by protecting the entire functional unit: preserving tonic dopamine release (presynaptic pathway) and, consequently, preventing stress signaling that triggers receptor overexpression (postsynaptic pathway). This synaptic stabilization positions VPA as a promising candidate not only for delaying motor progression but also for prolonging the therapeutic window of drugs such as L-DOPA, minimizing their long-term side effects.

3.4. Limitations and Perspectives

We acknowledge that this study did not directly quantify HDAC inhibition or GABAergic currents. The protective effect of VPA is likely multifactorial, involving both the suppression of pro-inflammatory cytokines and the enhancement of GABAergic tone, thereby reducing excitotoxicity. Furthermore, although the use of male rats allowed for control of hormonal variability, future studies should evaluate these effects in females to consider the neuroprotective role of estrogens.

Although our results show robust functional and biochemical recovery, because HPLC-EC measurement of DA levels is a validated surrogate for nigrostriatal integrity [23], we recognize that a limitation of this work is the lack of direct histological analysis by immunohistochemistry for tyrosine hydroxylase. Although DA levels quantified by HPLC-EC are a reliable indicator of presynaptic terminal integrity, evaluation of the number of neuronal bodies in the substantia nigra pars compacta would confirm whether the effect of VPA acts as a neuroprotector (cell survival) or as a neuromodulator (improvement of metabolic function). Future research should use stereological counts of TH+ neurons to corroborate the physical preservation of the nigrostriatal pathway.

4. Materials and Methods

4.1. Animals and Ethics

Adult male Wistar rats (270–290 g, N = 24) were housed under standard conditions (12:12 h light/dark cycle, 22–24°C). All protocols complied with the “Guide for the Care and Use of Laboratory Animals” [24] and NOM-062-ZOO-1999 and were approved by the INR-LGII Animal Care Committee (Protocol Approval Code: 98/22, Date: 14 November 2022).

4.2. Experimental Design

Rats were randomized into four groups (n = 6/group): SHAM Group: Vehicle treatment + Sham surgery; 6-OHDA Group: Vehicle treatment + 6-OHDA lesion; VPA-200 Group: VPA (200 mg/kg, p.o.) + 6-OHDA lesion; VPA-400 Group: VPA (400 mg/kg, p.o.) + 6-OHDA lesion. Valproic acid sodium salt (VPA; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile saline and administered daily by oral gavage at doses of 200 or 400 mg/kg for 20 consecutive days prior to stereotaxic surgery. Male rats were chosen to eliminate the neurochemical variability introduced by the estrogen cycle. Estradiol has been shown to have intrinsic neuroprotective effects on the dopaminergic system and modulates the density of D2 receptors [25,26,27].

4.3. Stereotaxic Surgery (6-OHDA Lesion)

Under ketamine/xylazine anesthesia (70/15 mg/kg i.p.), rats were placed in a stereotaxic frame. 6-hydroxydopamine (8 µg in 2 µL with 1% ascorbic acid) was infused unilaterally into the medial forebrain bundle SNpc. These coordinates (AP: −5.0 mm, ML: −2.0 mm, DV: −7.5 mm), frequently cited in comparative studies, consistently yield DA depletion exceeding 90%, making them a reliable choice for replicating this model [28]. Lesion efficacy was confirmed via apomorphine-induced rotation testing (0.5 mg/kg i.p.) on day 20 post-lesion; only rats exhibiting >7 full ipsilateral rotations per minute were included, as this indicates DA depletion of >90% [29].

4.4. Behavioral Assays

Beam Balance Test: Motor coordination and balance were assessed using a validated 0–6 scale over 3 days post-surgery, with a score of 6 indicating optimal performance [30].

Open Field Test: An exploratory activity was recorded for 5 min on day 20 using video tracking to measure center time (a marker of anxiolytic/exploratory behavior) versus periphery/corner time [31].

4.5. HPLC-EC Quantification of Dopamine

The striatum and motor cortex were rapidly dissected and homogenized in perchloric acid. Dopamine content was quantified using high-performance liquid chromatography with electrochemical detection (HPLC-EC), a highly sensitive technique for monoamine measurement [23].

4.6. Western Blot Analysis for D2 Receptors

To evaluate postsynaptic integrity and plasticity, striatal tissue was homogenized in RIPA buffer supplemented with a complete protease inhibitor cocktail. Total protein concentration was determined via the Bradford assay. Equal amounts of protein (30 µg) were separated by SDS-PAGE (10% gel) and transferred onto PVDF membranes. Membranes were blocked and incubated overnight at 4°C with primary antibodies: anti-dopamine D2 receptor (1:1000; Millipore, México City, Mexico, Cat# AB5085P) and anti-beta-actin (1:5000; Sigma-Aldrich, Cat# A5441) as a loading control [32]. Following incubation with HRP-conjugated secondary antibodies, protein bands were visualized using enhanced chemiluminescence (ECL). Densitometric analysis was performed using ImageJ 1.53t, with D2R intensity normalized to beta-actin. For densitometric quantification of the D2 receptor, the analysis focused exclusively on the main band corresponding to the molecular weight of the native receptor (~50 kDa). In some samples from the 6-OHDA group, an additional band with a higher molecular weight was observed; however, to ensure technical consistency and avoid possible biases derived from protein aggregates or changes in post-lesion glycosylation, this signal was excluded from the statistical analysis, following previously reported standardization criteria.

4.7. Statistical Analysis

Data are presented as mean +\ SEM. A one-way ANOVA followed by Tukey’s post hoc test was used for multiple-group comparisons. A two-tailed value of * p < 0.05 was considered statistically significant.

5. Conclusions

This study establishes that valproic acid at 400 mg/kg provides profound, dose-dependent protection against nigrostriatal degeneration in a rat model of Parkinson’s disease. This protection is evidenced by the possible preservation of the motor function and striatal dopamine levels, as well as by the stabilization of striatal dopamine D2 receptor expression, which prevents pathological denervation supersensitivity. These findings support the potential of high-dose VPA as a promising disease-modifying adjuvant therapy to delay PD progression and mitigate the development of long-term motor complications.