SL-327

Effects of ERK1/2 kinases inactivation on the nigrostriatal system of Krushinsky-Molodkina rats genetically prone to audiogenic seizures

Nadezhda A. Dorofeeva, Yuliya S. Grigorieva , Liubov S. Nikitina, Elena
A. Lavrova, Elizaveta V. Nasluzova, Margarita V. Glazova & Elena V. Chernigovskaya

To cite this article: Nadezhda A. Dorofeeva, Yuliya S. Grigorieva , Liubov S. Nikitina, Elena A. Lavrova, Elizaveta V. Nasluzova, Margarita V. Glazova & Elena V. Chernigovskaya (2017): Effects of ERK1/2 kinases inactivation on the nigrostriatal system of Krushinsky-Molodkina rats genetically prone to audiogenic seizures, Neurological Research, DOI: 10.1080/01616412.2017.1356156
To link to this article: http://dx.doi.org/10.1080/01616412.2017.1356156

Introduction

A hierarchical organization of brain structures involved in the regulation of motor activity are complex, where coordinated activity plays a key role in realization of motor response after obtaining sensorimotor, emotional, or cognitive information [1,2]. Different parts of the brain are differentially involved in the expression of seizures [3]. It was demonstrated in the genetic models of audio- genic epilepsy that the primary locus of the generation of convulsive seizure caused by sound is located in the brain stem [4]. At the same time, basal ganglia complex representing various subcortical cell groups plays a sig- nificant role in the regulation of motor activity, including epileptiform seizures [5]. The central element of basal ganglia is the corpus striatum, which collects and ana- lyzes information coming from other parts of the brain, such as cortex and thalamus [6].

The 95% of cells of the striatum are GABAergic neurons. Two subpopulations of long axon moderately spiny GABAergic neurons are distinguished that differ in the directionality of efferent projections and in the composition of expressed pro- teins. The first subpopulation of neurons innervates the reticular part of the substantia nigra. They are charac- terized by the expression of dopamine D1 receptor and gives rise to a pro-convulsive direct path of the basal ganglia. The second subpopulation directs processes to the globus pallidus, expresses a dopamine D2 receptor and gives rise to indirect anti-convulsive path of the basal ganglia [7,8].

It was shown that ERK1/2 kinases play an impor- tant role in the development of seizure activity [9–13]. Stimulation of the cortico–striatal projections causes the activation of the phosphorylation of ERK1/2 kinases in striatal neurons [14,15], and it is known that these kinases are interacting with the dopamine and gluta- mate receptors. This receptor-dependent interaction determines the excitatory or inhibitory response of the striatum to the different stimuli [8,16,17]. Glutamate can exert activating effect on ERK1/2 kinase by binding with all receptors types (NMDA, AMPA, and kainate). On the other hand, an activation of ERK1/2 kinases leads to an increase in the NR2B subunit of NMDA receptors [9]. The activity of ERK1/2 kinases itself is increased upon activation of dopamine D1 receptors in CNS neurons including the striatum [18–21]. Moreover, in mice with NR2B mutation, which prevent its phosphorylation, binding the D1 receptors or administration of dopamine did not result in significant activation of ERK1/2 [18]. These data allow us to consider ERK1/2 as a key mediator that could coordinate the effect of dopaminergic and glutamatergic systems on the activity of GABAergic neurons in the striatum [10,22].

At present, a possibility of using the pharmacological properties of ERK1/2 is considered [9,23]. One potential approach in the treatment of some diseases is to reduce the activity of ERK1/2 kinases by the selective blocker SL327. SL327 penetrates the blood–brain barrier [24,25], which allows intraperitoneal injections with minimal stress to the animal. Previously, the important role of ERK1/2 kinases in the formation of epileptiform activity in various models of epilepsy using SL327 was shown [11,26,27]. In this work, we examine the nigrostriatal system of the KM rats genetically prone to audiogenic seizure (AGS), which were injected with the SL327 and subjected to the sound stimulation.

Material and methods

Animals

Adult 4 months old, 250–300 g Krushinsky-Molodkina rats (KM) (Moscow State University, Russia), an audio- genic rat strain [28], were used in the experiments. These rats display stable sound-induced clonic–tonic seizures [28], thus presenting a well-established animal model of epilepsy. Two weeks before experiments rats were tested by the audio stimulation to check AGS expression.The animals were housed in individual cages under natural light–dark cycle with free access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee at the Sechenov Institute of Evolutionary Physiology and Biochemistry.

SL 327 treatment

Inhibitor of MEK1/2 activity SL 327 (Tocris Bioscience, Bristol, U.K.) was used to block ERK1/2 activation [25]. Animals were i.p. injected, the control group (4 males and 5 females) with a single dose of vehicle (dimethyl sulfoxide, DMSO), and SL 327 group (1 male and 8 females) was treated with 50 mg/kg SL 327 dissolved in DMSO. The injections were done 60 min before audio stimulation.

Induction of audiogenic seizure

The AGS were induced by the sound stimulation at 10 kHz and 50 dB generated by an electronic device [26,29]. Every animal was placed in testing chamber, and then exposed to the acoustic stimuli for seizure genera- tion. Before testing, a 15-min quiet period was allowed for the animal to acclimate to the observation cham- ber. Seizures in KM rats start after short latent period (2–10 s) followed by wild running, clonic and then tonic phases that finalize with ataxia. The sound lasted until the appearance of wild running behavior, but no longer than 90 s, if seizure was absent. All experiments were video-recorded for further behavior analysis.

Seizure analysis

We analyzed the duration of the latency period (the time between the start of sound stimuli and the beginning of seizure activity), and all successive stages – wild run- ning, clonus, and tonus. To analyze the severity of sei- zure, the following classification was used as the scores: 0 – absence of seizure; 1 – oral-facial seizure; 2 – wild running only; 3 – wild running and clonic phase devel- opment; 4 – seizure with incomplete tonic phase; 5 – complete seizure.

Sample preparation

After audiogenic stimulation the control rats were decap- itated at ataxia, SL 327 treated rats were decapitated after 90 s of sound stimulation or at ataxia if the seizure had started. One half of the brains from all animals were fixed in 4% paraformaldehyde for morphological and immunohistochemical assays. The second parts were dissected for the striatum and substantia nigra, and these zones were homogenized separately for further biochemical analysis.

Western Blot Analysis

Tissues were homogenized in lysis buffer containing protease inhibitors and phosphatase inhibitor cocktail (both from Sigma-Aldrich, St. Louis, MO, U.S.A.). The total protein concentrations were determined by Bio- Rad protein assay (Bio-Rad Laboratories Inc., Hercules, CA, U.S.A.). Equal amounts of protein (15 μg per line) in sample buffer (Bio-Rad) were denatured at 95 °C for 5 min and separated on 10% acrylamide gel. The pro- teins from the gel were transferred to a nitrocellulose membrane (Santa Cruse). The membranes were incu- bated in 3% non-fat dry milk in Tris buffer (0.1% Tween 20, 0.2 mM Tris, 137 mM NaCl) for 30 min and then incubated overnight with primary antibodies against: p-ERK1/2 (Thr202/Tyr204; 1:1000; Cell Signaling Technology, Beverly, MA, U.S.A.); p-synapsin I (Ser62, 67, 1:500; ProSci Incorporated, Poway, CA, U.S.A.);synapsin I (1:3000, Millipore, Temecula, CA, U.S.A.); GAD65 (1:2000; Abcam plc, Cambridge, U.K.); GAD67 (1:2000; Millipore); TH (1:1000, Sigma-Aldrich); p-TH (Ser31, 1:1000, Millipore); D1a receptor (1:200, Millipore); D2 receptor (1:500, Millipore); actin (1:1000; Abcam). Subsequently, the membranes were incubated with secondary anti-rabbit (1:8000; Sigma-Aldrich) or anti-mouse (1:8000; Sigma-Aldrich), followed by chemiluminescent detection by ECL-plus (Amersham, GE Healthcare, Little Chalfont, Buckinghamshire, U.K.).

Immunohistochemistry

The brain sections were incubated with primary anti- bodies against: VGLUT2 (1: 200; Abcam); NR2B (1:500, Abcam plc, Cambridge, U.K.); p-TH (Ser31) (1:1000, Millipore), and then with biotinylated secondary anti- bodies (Vector Labs) and streptavidin–peroxidase com- plex (Sigma-Aldrich). The peroxidase reaction of the avidin–biotin complex was revealed in the buffer con- taining 3,3′ diaminobenzidine (DAB; 0.05%) and hydro- gen peroxide (0.01%). Additionally, reactions lacking primary antibodies were done to ensure the specificity of the observed staining.

Evaluation of sections and statistical analysis

The sections were processed under standardized condi- tions in every experiment, i.e. control and experimental groups in each experiment were collected, fixed, and then processed for immunohistochemistry simultane- ously. Keeping this order for the samples preparation allowed semi-quantitative analysis of protein amount in the histological slices by measurement of optical density [30]. Five sections at the same level of the studied zones were analyzed from each animal. The relative optical density of immunopositive substances in the striatum and substantia nigra was estimated, and average and standard deviation were calculated. Optical density reflecting the content of immunopositive substance was calculated as the ‘‘grey level’’ (GL) of immunoreactive field of tissue minus background GL. Optical density of the background was estimated at the same slice in non-immunoreactive brain tissue field. Results are pre- sented in relative units of optical density.

Statistical analysis was carried out by the Student’s t test, and values are expressed as mean ± SE for immuno- histochemistry, and for Western blot analysis. P values of ≤0.05 were used for statistical significance.

Results

Behavior testing

Completed behavior analysis was presented in our previ- ous work [26]. In brief, all control animals demonstrated complete AGS (N = 9). After SL 327 injections (N = 9) AGS was completely absent in five rats, one rat lacked tonic and clonic phases and one rat expressed only clonic phase; and the remaining two animals demonstrated complete seizures. The data analysis for protein expres- sion in the striatum and substantia nirga was done in KM rats at ataxia stage (control animals) and in rats, which do not express AGS after SL327 injections (N = 5).

Inhibition of ERK1/2 activity leads to de- phosphorylation of Synapsin I, accumulation of VGLUT2 and decreasing of NR2B in the striatum

The SL327 administration caused a decrease in the con- tent of phospho-ERK1/2 kinases both in the striatum and in the substantia nigra (data not shown) in the same manner as it was demonstrates for the hippocampus and the cortex [26]. Then we analyzed phosphorylation level of Synapsin I at Ser62/67, which are the target for ERK1/2 [31]. The results showed decreasing of Synapsin I phosphorylation (Figure 1(A)), which probably can lead to disruption of exocytosis. On the other hand, we observed an increase in the content of the vesicular glu- tamate transporter 2 (VGLUT2) in the fibers of the stri- atum (Figure 1(B)–(D)). Obtained results demonstrated that inactivation of the ERK1/2 leads to a disturbance of the glutamate release, thereby causing accumulation of the glutamate in presynaptic terminals of the stria- tum fibers. Also in the striatum, we observed significant reduction of NR2B positive cells after SL 327 injections (Figure 1(E)–(G)).

SL327 injections changes the activity of GABAergic neurons in the nigrostriatal system

Using Western blot analysis, we demonstrated significant decrease in GAD65 (Figure 2(A)) and GAD67 (Figure 2(B)) in the dorsolateral striatum in the KM rats, which were injected with SL327. This indicates a decline in the synthesis of GABA in this brain structure and suggests a decrease in GABAergic innervation of the globus pal- lidus and the substantia nigra.

On the other hand, we showed an increased level of the GAD65 (Figure 2(C)) and the GAD67 (Figure 2(D)) in the substantia nigra of SL327 injected KM in comparison with control animals. The increased activity of the GABAergic system of the reticular part of the substantia nigra could lead to increased inhibitory effect on the thalamus, which sends glutamatergic projections to the cortex and to the brain stem, thereby reducing seizure expression.

Changes in the activity of dopaminergic neurons in the nigrostriatal system

Using immunohistochemistry, we showed significant reduction in immunoreactivity of tyrosine hydroxylase (TH) phosphorylated at the Ser-31 site in the compact part of the substantia nigra (Figure 3(A)–(C)). In the stri- atum, by Western blot analysis, we also observed decreas- ing of TH phosphorylation after inactivation of ERK1/2 kinases (Figure 3(D), (E)). In addition, the level of unphosphorylated TH protein was also reduced (Figure 3(D), (F)). Our data revealed a significant decrease in the activity of the dopaminergic system by the SL327.

Figure 1. Inhibition of ERK1/2 activity leads to alteration in synapsin I, VGLUT2, and NR2B in the striatum. A – The level of Synapsin I phosphorylation level was estimated by Western blot. The protein extracts of the striatum (N = 6 for control and N = 5 for SL 327 treated group) were immunoblotted with affinity purified antibodies against p-synapsin I (Ser62/67). B, C, D – Immunodetection of VGLUT2 in control KM rats (B) and SL-327 treaded rats (C). Evaluation of the optical density demonstrated significant increase in VGLUT2 in the striatum of SL-237 injected KM rats (D). Optical density presented in arbitrary units. Axis y: optical density (arbitrary units). Data are shown as mean ± SE. *P < 0.05 vs. control. E, F, G – Immnunohistochemical detection of NR2B subunit of NMDA glutamate receptor in the striatum of control KM rats (E) and SL327 injected rats (F). Arrows point at positive cells. And the number of NR2B immunopositive neurons was counted in the striatum (G). Data are shown as mean ± SE. *P < 0.05 vs. control. For the evaluation of optical density and counting of immunopositive cells, five sections from each animal (N = 6 for control and N = 5 for SL 327 treated group) were analyzed.

Figure 2. Analysis the expression of GAD65 and GAD 67 in the striatum and substantia nigra. A, B – Western blot analysis demonstrated that content of GAD65 (A) GAD67 (B) was significantly diminished in the striatum. While in the substantia nigra, these enzymes for GABA synthesis were increased (C, D) (N = 6 for control and N = 5 for SL 327 treated group).Notes: Optical density presented in arbitrary units. Axis y: optical density (arbitrary units). Data are shown as means ± SE. *P < 0.05 vs. control.

We also noticed the decrease in the content of D1 receptor in the striatum (Figure 4(A)), which can lead to a decrease in the density of GABAergic innervation of the substantia nigra. Moreover, the reduction of immu- noreactivity of the dopamine D2 receptors was also observed after ERK1/2 inactivation (Figure 4(B)), that could lead to an increasing of GABA release in the outer segment of the globus pallidus and, in turn, finalized with a reduction of inhibitory impact on GABAergic neurons in the reticular part of the substantia nigra.

Discussion

In the present study, we evaluated the changes in the activity of the nigrosriatal system in KM rats, which demonstrated the total blockade of AGS after inhibition of ERK1/2 activity by SL327 injection. We found that such inhibition increases the amount of VGLUT2 and decreases Synapsin I phosphorylation in the dorsolateral part of the striatum compared with the control KM rats. In our previous study, we showed that inactivation of ERK1/2 kinases by SL327 decreased Synapsin I activ- ity in the hippocampus and cortex, which also led to accumulation the VGLUT2 [26]. We propose that dys- regulation of exocytosis by low activity of ERK1/2 results in an accumulation of glutamate in the terminal part of the motor cortex neurons innervating GABAergic neu- rons in the striatum thus leading to a decreased syn- thesis of GABA in the striatum. At the same time, we observed reduction in the content of VGLUT2 upon the inactivation of ERK1/2 kinases in the inferior colliculus (unpublished data). Thus, decrease in the activity of ERK1/2 kinases in different brain regions may lead to the opposite effects.

It is known that NMDA receptors tightly participate in epileptogenesis [32–34] and even considered as a therapeutic target by anti-epileptic therapy [35,36]. In transgenic mice with overexpression of MEK1, which are predisposed to epileptic seizures, an increased level of the NR2B at the post-transcriptional level without affecting the level of the mRNA was reported [9]. We observed low levels of the NR2B subunit of NMDA receptors in the hippocampus and in the inferior col- liculus in naïve KM rats compared to Wistar rats while ERK1/2 activity was increased (unpublished data). Here we demonstrated that inhibition of ERK1/2 activity leads to decrease in the number of cells producing NR2B in the striatum. We suggest that inactivation of ERK1/2 decreases NR2B synthesis in GABAergic neurons of the striatum. Moreover, this data combined with a decrease in exocytosis of glutamate lowers glutamatergic input from neurons of the motor cortex on GABAergic neu- rons of the striatum. That in turn reduces synthesis of GABA – GAD65 and GAD67 in the striatum. On the other hand changes in the level of glutamate decar- boxylases may also be the result of the activity of the dopaminergic system. We showed a decreased phos- phorylation level of TH at Ser-31 site, which is a spe- cific site for ERK1/2 [37], after SL327 treatment that, respectively, can lead to the reduction in the activity of the nigrostriatal dopaminergic neuronal system [38,39]. It has previously been shown that the haloperidol- dependent increased intensity of phosphorylation of TH was reduced by SL327 injection in the striatum of mice [40], which correlates with our results. Low dopamin- ergic activity leads to the decrease in the expression of the D1 and the D2 dopamine receptors in the striatum of KM rats. Obviously, D1 receptor reduction decreased the “direct” pathway input onto GABAergic neurons in the reticular part of the substantia nigra, resulting in activation of these neurons. Low level of D2 receptor leads to activation of “indirect” way of regulation of GABAergic neurons in the reticular part of the substan- tia nigra [10,41]. We show here that an activation of the D2-dependent pathway, caused by the SL327 injection, could lead to an increasing of glutamate production in the neurons of the subthalamic nucleus, which sends projections to GABAergic neurons in the reticular part of the substantia nigra [42]. Indeed, we observed signif- icantly upregulated level of VGLUT2 after inhibition of ERK1/2 in the substantia nigra of KM rats. Additionally, we have shown an increased level of both GAD65 and GAD67 in the reticular part of the substantia nigra. We therefore suggest that low activity of the D1-dependent pathway and high activity the D2-dependent pathway could result in the activation of GABAergic neurons in the reticular part of the substantia nigra [43–45].

Figure 3. SL-327 treatment decreases tyrosine hydroxylase in the substantia nigra and striatum. A, B, C – Immunostaining for p-TH in the substantia nigra of control (A) and SL-327 (B) injected KM rats. Arrows point at positive cells. Evaluation of the optical density revealed significant decreasing of p-TH content in KM rats after inhibition of ERK1/2 activity (C). D, E, F – the level of p-TH and TH was estimated by Western blot analysis in the striatum (D). Obtained data revealed decreasing in TH phosphorylation at Ser31 (E) as well as in TH content (F) after ERK1/2 inhibition.
Notes: Optical density presented in arbitrary units. Axis y: optical density (arbitrary units). Data are shown as means ± SE. *P < 0.05 vs. control. For the evaluation of optical density, five sections from each animal (N = 6 for control and N = 5 for SL 327 treated group) were analyzed.

Figure 4. Inhibition of ERK1/2 activity decreases of D1 and D2 dopamine receptor content in the striatum. A, B – Western blot analysis demonstrated significantly decreased level of D1 (A) and D2 (B) receptor expression in KM rats injected by SL-327 (N = 6 for control and N = 5 for SL 327 treated group). Notes: Optical density presented in arbitrary units. Axis y: optical density (arbitrary units). Data are shown as means ± SE. *P < 0.05 vs. control.

Here we demonstrated that blockade of AGS by inactivation of ERK1/2 inhibits Synapsin I activity and glutamate release in the striatum, decreases the activity of dopaminergic neurons, and leads to upregulation of GABA synthesis in the substantia nigra. We suggest that nigrastriatal system may be involved in or affected by the AGS expression in KM rats.

Acknowledgment

Part of the analysis was done at Research Resource Center for the physiological, biochemical and molecular-biological research #441590 at Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences.

Disclosure statement

The authors declare that they have no conflict of interest.

Funding

This work was supported in part by Russian Foundation for Basic Research [grant number RFBR 11-04-00648, grant number 16-04-00681].

ORCID

Yuliya S. Grigorieva http://orcid.org/0000-0001-8929-0523

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