Neuroscience Letters 586 (2015) 24–30

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Regulation of dopamine D2 receptor-mediated extracellular signal-regulated kinase signaling and spine formation by GABAA receptors in hippocampal neurons Dong-Hoon Yoon a,1 , Sehyoun Yoon a,1 , Donghoon Kim a , Hyun Kim b , Ja-Hyun Baik a,∗ a b

Molecular Neurobiology Laboratory, Department of Life Sciences, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea Department of Anatomy, College of Medicine, Korea University, Brain Korea 21, Seoul 136-705, South Korea

h i g h l i g h t s • • • • •

In hippocampal neurons, dopamine D2 receptors (D2Rs) interact with GABAA R. GABAA R negatively modulate D2R-induced ERK activation in hippocampal neurons. D2R-mediated ERK phosphorylation controls spine formation in hippocampal neurons. GABAA R negatively regulate D2R-induced spine formation through ERK signaling. D2R-GABAA R can regulate the hippocampal synaptic plasticity through ERK signaling.

a r t i c l e

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Article history: Received 18 September 2014 Received in revised form 12 November 2014 Accepted 2 December 2014 Available online 4 December 2014 Keywords: Dopamine GABA ERK Hippocampal neurons Dendritic spine

a b s t r a c t Dopamine (DA) signaling via DA receptors is known to control hippocampal activity that contributes to learning, memory, and synaptic plasticity. In primary hippocampal neuronal culture, we observed that dopamine D2 receptors (D2R) co-localized with certain subtypes of GABAA receptors, namely ␣1, ␤3, and ␥2 subunits, as revealed by double immunofluorocytochemical analysis. Treatment with the D2R agonist, quinpirole, was shown to elicit an increase in phosphorylation of extracellular signal-regulated kinase (ERK) in hippocampal neurons. This phosphorylation was inhibited by pretreatment with the GABAA receptor agonist, muscimol. Furthermore, treatment of hippocampal neurons with quinpirole increased the dendritic spine density and this regulation was totally blocked by pretreatment with a MAP kinase kinase (MEK) inhibitor (PD98059), D2R antagonist (haloperidol), or by the GABAA receptor agonist, muscimol. These results suggest that D2R-mediated ERK phosphorylation can control spine formation and that the GABAA receptor negatively regulates the D2R-induced spine formation through ERK signaling in hippocampal neurons, thus indicating a potential role of D2R in the control of hippocampal neuronal excitability. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Dopamine (DA) is a predominant catecholamine neurotransmitter in the mammalian brain where it controls a variety of functions, including locomotor activity, hormone secretion, and reward-related behaviors [1,2]. DA is synthesized by mesencephalic neurons in the substantia nigra (SN) and ventral tegmental area and these DA neurons project to the striatum, cortex, and

∗ Corresponding author. Tel.: +82 2 3290-3455; fax: +82 2 927-9028. E-mail address: [email protected] (J.-H. Baik). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2014.12.010 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.

limbic systems including the hippocampus, amygdala, and nucleus accumbens. DA D2 receptors (D2Rs) are one of the predominant DA receptor subtypes abundantly expressed in the brain. D2Rs are coupled to G␣i and G␣o proteins and negatively regulate the production of cAMP, resulting in decreased PKA activity, activation of K+ channels, and the modulation of numerous other ion channels [1–4]. Absence of D2R, as revealed by D2R knockout mice, leads to animals with impaired locomotion and coordination of movement [5], but also altered synaptic plasticity [6] and hormonal dysfunction [7,8]. As a modulatory neurotransmitter, DA can modulate excitatory and inhibitory synaptic transmission in different brain areas, depending on the receptor type and probably also upon the

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signaling pathway exploited. The dopaminergic regulation of neuronal excitability appears to occur in a variety of ways. It has been reported that dopamine D5 and GABAA receptors are capable of forming a receptor complex through direct protein–protein interactions and these receptor interactions mutually inhibit the activities of both receptors [9]. These interactions between different receptors can contribute to modulation of output of the neural circuit, thus alteration of this interaction could be an important parameter underlying the pathophysiology of DA neurotransmission systems. We have previously shown that in mice lacking D2R, the level of glutamic acid decarboxylase, an enzyme involved in GABA synthesis, was much higher than in wild-type mice and the absence of D2R alters GABAergic neurotransmission, specifically on GABAA receptor-mediated signaling [5,10]. These findings suggest that D2R-GABAA receptor interactions may contribute to pathological mechanisms in brain networking. Further investigations of this interaction will increase our understanding of DAergic disorders, including potential involvement of the GABA system. In the present study, we examined whether D2R can interact with the GABAA receptor in hippocampal neurons and whether the GABAA receptor can regulate D2R-mediated signaling, for example, D2R-induced activation of extracellular signal-regulated kinase (ERK), which is a well-known signal pathway of the D2R [11–14]. Our results show that D2R-mediated ERK activation in hippocampal neurons contributes to dendritic spine morphogenesis and that the GABAA receptor negatively regulates the D2R-mediated signaling, thus indicating a possible role of DA-GABA interaction in control of hippocampal neuronal excitability.

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(10 ␮M), the neurons were washed with ice-cold PBS and lysed in a cell lysis buffer for 10 min on ice followed by centrifugation at 12,000 rpm for 30 min at 4 ◦ C. Protein (about 40 ␮g) was separated on a 10% SDS-PAGE and blotted onto pre-wetted polyvinylidene difluoride nitrocellulose membrane. The membrane was incubated with a mouse monoclonal anti-p-ERK (Cell Signaling, 1:3000) or a rabbit monoclonal anti-ERK (Santa Cruz, 1:4000). Specific bands were detected by enhanced chemiluminescence (ECL, Amersham Biosciences) and analyzed using an LAS3000 image analysis system (Fuji). 2.4. PM-YFP transfection and pharmacological treatments Hippocampal neurons were transfected at DIV 14 with plasma membrane-targeted yellow fluorescent protein (PM-YFP) [15] by the calcium phosphate method. Before transfection, media were changed with fresh NB media containing B27 supplement, Glutamax I, and 5 mM MgCl2 for 5 × 104 cells on an 18 × 18 mm coverslips. The Ca2+ -phosphate transfection protocol was followed according to Min Jian & Gong Chen [16]. After treatment with muscimol or quinpirole in the presence or absence of pretreatment with PD98059 (50 ␮M for 30 min) or haloperidol (10 ␮M for 10 min) or muscimol (10 ␮M for 10 min), hippocampal neurons were fixed using prepared 4% paraformaldehyde in PBS for 15 min at 4 ◦ C. Non-specific binding was reduced by 30 min incubation in PBS containing 5% FBS and 0.2% Triton X-100. Enhanced yellow fluorescent protein (eYFP) signal was amplified by immunocytochemistry. The samples were incubated with anti-GFP (Santa Cruz, sc-9665) in blocking buffer overnight at 4 ◦ C and detected using anti-rabbit Alexa 488 IgG.

2. Material and methods 2.5. Image analysis of dendritic spine morphology 2.1. Primary neuronal cell culture Female ICR mice were purchased from the Charles River Laboratories. Pregnant mothers were sacrificed at embryo day 18 (E18) for hippocampal neuronal cultures or at E13 for mesencephalic neuronal cultures. To prepare primary neuronal cultures, the hippocampus or the mesencephalon was dissected from 18day or 13-day gestation mouse embryos and primary neuronal cultures were prepared as described previously [12–14]. Neurons were maintained at 37 ◦ C in a humidified with 5% CO2 atmosphere in neurobasal media supplemented with B27 (Invitrogen) and GlutaMax-1 for 14 days (hippocampal neurons) or 3 days (mesencephalic neurons). 2.2. Immunocytochemistry For dual fluorescent staining, hippocampal neurons were fixed using freshly prepared 4% paraformaldehyde in PBS for 15 min at 4 ◦ C. Nonspecific binding was reduced by a 30 min incubation in PBS containing 5% FBS and 0.2% Triton X-100. Cells were incubated with specific antibodies for D2R (Chemicon, AB5084P, 1:200), GABAA receptor ␣1 subunit (Santa Cruz, sc-7348, 1:200), ␤3 subunit (Chemicon, MAP341, 1:200), or ␥2 subunit (Santa Cruz, sc-7371, 1:200) overnight at 4 ◦ C. After washes with PBS containing 0.2% Triton X-100, samples were incubated at RT for 1 h with Alexa Fluor 488 donkey anti-rabbit IgG, Alexa Fluor 568 donkey anti-mouse IgG or Alexa Fluor 568 donkey anti-goat IgG (Invitrogen, 1:200). Images were taken by Zeiss LSM 700 META laser scanning microscope (Carl Zeiss) with a 63× oil immersion objective lens. 2.3. Western blot analysis of p-ERK After treatment with quinpirole (10 ␮M) in the presence or absence of pretreatment with bicuculline (10 ␮M) or muscimol

Images (2048 × 2048 resolution) were acquired using a Zeiss LSM 700 META laser scanning microscope (Carl Zeiss) with a 40× water immersion objective lens. For higher magnification pictures, a 63× oil immersion objective lens was used. Transfected neurons were randomly selected for quantification and spine density was measured by counting the number of protrusions on 21 to 33 neurons in each condition. Spine densities were estimated by counting the number of spines along 200 ␮m segments of a primary dendrite. Densities were expressed as spines per 50 ␮m. The primary dendrite was identified as the thickest and longest dendrite among dendrites which projected from soma. The length of the primary dendrite over 100 ␮m was used for analysis. For analysis of spine density, protrusions of up to 4 ␮m of the length of spines were considered. Dendrites were manually traced and spine length was measured with the DP72 Imaging System (Olympus). 2.6. Statistical analysis For statistical analyses, data were analyzed with multiple comparisons using one-way ANOVA followed by the Student–Newman–Keuls’s post hoc test. 3. Results 3.1. Hippocampal neurons co-express D2Rs and GABAA receptors It has been reported that hippocampal neuronal cells express D2Rs [17,18] and also express GABAA receptor subunits [19]. We investigated the expression of D2R and GABAA receptors in primary hippocampal neuronal cultures. Dissected hippocampal neuronal cells from embryo day 18 (E18) ICR mice were cultured and at 14 days in vitro, neurons were immunostained with anti-D2R and antiGABAA receptor subunits ␣1, ␤3, or ␥2. The localization of D2R

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Fig. 1. Hippocampal neurons co-express D2R and subunits of GABAA receptors. Immuno-cytochemical images of D2R staining (green) with GABAA R ␣1 subunit (A), or GABAA R ␤3 subunit (B), or GABAA ␥2 subunit (C), (red) are shown. Co-localization of D2R immunoreactivity with GABAA subunits is marked by a solid arrow. D2R only-expressing neurons are marked by an empty arrow. Scale bar, 20 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

and subunits of GABAA receptor was determined by confocal fluorescence microscopy and images were merged to determine the colocalization of these proteins (Fig. 1). We observed that D2Rpositive neurons are colocalized with GABAA receptor subunits ␣1 (Fig. 1A), ␤3 (Fig. 1 B), and also ␥2 (Fig. 1C), as evidenced by double immunofluorocytochemical analysis coupled with confocal microscopy. 3.2. GABAA receptors regulate D2R-mediated ERK activation in hippocampal neurons We previously reported that stimulation of the D2R can induce extracellular signal-related kinase (ERK) activation [12–14] which may play a crucial role in the development of dopaminergic neuron regulation by not only increasing the number of DA neurons but also the number and length of neurites. We tested whether the stimulation of D2Rs can induce ERK activation in hippocampal neurons. Treatment with quinpirole, a D2R agonist, increased ERK phosphorylation in hippocampal cultures (Fig. 2A). We also exam-

ined whether a GABAA receptor agonist or antagonist could regulate ERK activation in hippocampal cultured cells. Treatment with muscimol, a GABAA receptor agonist, had no effect in hippocampal neuronal cells, while the GABAA receptor antagonist, bicuculline, induced an increase in ERK phosphorylation. Interestingly, pretreatment with muscimol before quinpirole treatment significantly blocked quinpirole-induced phosphorylation of ERK in hippocampal neurons (Fig. 2A). However, pretreatment with bicuculline before quinpirole treatment did not affect ERK phosphorylation induced by quinpirole in hippocampal neurons (Fig. 2). Therefore, activation of the GABAA receptor inhibits the D2R-mediated ERK activation in hippocampal neurons. We previously demonstrated that D2R-mediated ERK activation plays a critical role in development of mesencephalic DA neurons. Here we examined the effect of a GABAA receptor agonist and an antagonist in mesencephalic neuronal cultures. In mesencephalic neurons, treatment with the D2R agonist, quinpirole, induced the activation of ERK signaling as previously observed [12–14]. Muscimol pretreatment did not affect the quinpirole-induced ERK

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Fig. 2. Regulation of D2R-mediated ERK activation by GABAA receptors in hippocampal neurons. (A) Hippocampal neuronal cells (DIV 14) were treated for 10 min with muscimol (10 ␮M), bicuculline (10 ␮M), or quinpirole (10 ␮M) with or without pretreatment of muscimol or bicuculline for 10 min. Drug effect F5,41 = 7.098. (B) Mesencephalic neuronal cells (DIV3) were treated using the same procedure as for hippocampal neurons. Drug effect F5,29 = 8.821. Representative Western blot and quantitative relative intensity analysis from Western blots of phospho-ERK and ERK levels are shown. The mean values ± SEM are shown. One-way ANOVA followed by a Newman–Keuls test for *p < 0.05, **p < 0.01, ***p < 0.001 control versus drug-treated cells; $$p < 0.01 quinpirole versus muscimol + quinpirole; ##p < 0.01 bicuculline + quinpirole versus bicuculline or quinpirole or muscimol + quinpirole.

activation, while bicuculline alone or bicuculline pretreatment prior to quinpirole induced a significant ERK activation (Fig. 2B). Therefore, D2R-induced ERK signaling is negatively regulated by GABAA receptors in the hippocampal neurons. 3.3. D2R-mediated spine morphogenesis is regulated by activation of GABAA receptors We then examined the physiological role of D2R-mediated ERK activation in hippocampal neurons. It has been reported that ERK activation is important for the formation of novel dendritic spines in hippocampal neurons [20,21]. We thus set out to analyze whether D2R-induced ERK activation can regulate the formation of novel dendritic spines in hippocampal neurons. Hippocampal neurons were treated with quinpirole for 1 h, 6 h, or 24 h. Spine densities were measured by counting the number of spines along 200 ␮m segments on a primary dendrite, considering up to 4 ␮m of the length of spines. Quinpirole treatment of hippocampal neuronal cultures significantly promoted spine density over the treatment period from 1 h to 24 h. In 16 DIV cultures, treatment with quinpirole for 1 h had a statistically significant effect (p < 0.05) increasing the density of spines from 8.70 ± 0.64 to 14.24 ± 0.84 per 50 ␮m of dendritic length (Fig. 3A). Pretreatment with the D2R antagonist, haloperidol, significantly reduced the quinpirole-induced increase in spine density in hippocampal neurons over a 1 h period to 8.77 ± 0.83 per 50 ␮m dendritic length (Fig. 3A). This suggests that the regulation of spine number is mediated by D2R-mediated signaling. Since stimulation of D2Rs employs ERK activation, we tested whether ERK signaling can regulate the formation of spines.

Pretreatment with the MEK (ERK kinase) inhibitor, PD98059, suppressed the quinpirole-induced increase in spine density in hippocampal neurons over a 1 h period, decreasing spine numbers to 7.61 ± 0.74 per 50 ␮m dendritic length (Fig. 3A). This indicates that the regulation of spine formation is mediated by D2R-induced ERK signaling. We further analyzed the effect of the GABAA receptor agonist, muscimol. Muscimol treatment alone displayed no effect on spine density. However, pretreatment with muscimol led to a complete inhibition of the quinpirole-induced increase in spine density in cultured hippocampal neurons over 1 h period to 9.45 ± 0.87 per 50 ␮m dendritic length (Fig. 3A). Spine analysis revealed that the effect obtained with D2R agonist and antagonist and the pretreatment with haloperidol, PD98059, or muscimol resulted in similar regulation of spine density at the 6 and 24 h time points as well (Fig. 3B and C). These results indicate that in hippocampal neurons, D2R-mediated ERK activation can promote spine morphogenesis and that activation of GABAA receptors could inhibit the increase of spine density induced by D2R activation. 4. Discussion Although dopaminergic signaling in the hippocampus is not well understood, increasing evidence suggests that dopamine plays an important role in the detection of novel information, modulation of memory formation, and synaptic plasticity [22–24]. In the present study, we found that in hippocampal neurons, D2Rs interact with GABAA receptors and that GABAA receptors negatively modulate D2R-induced ERK activation. Furthermore, we observed that D2R-mediated ERK phosphorylation can control

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Fig. 3. Regulation of the D2R-mediated spine formation by GABAA receptors in hippocampal neurons. Representative images of dendrite segments after treatment with each drug are shown. Spine densities on a primary dendrite were analyzed. The hippocampal neurons were dissected on E18 and PM-YFP transfection was conducted at DIV 14. All drug treatments were conducted at DIV15 for 1 h (A), 6 h (B), or 24 h (C). Drug effect 1 h (A) F5,157 = 9.482; 6 h (B) F5,93 = 3.582; 12 h (C) F5,130 = 5.618. Scale bars, 20 ␮m (upper), 2.5 ␮m (lower). The mean values ± SEM are shown (n = 21 − 33 per group). One-way ANOVA followed by a Newman–Keuls test for *p < 0.05, **p < 0.01, ***p < 0.001 control versus drug-treated cells; #p < 0.05, ##p < 0.01, ###p < 0.001 quinpirole versus PD98059 + quinpirole or haloperidol + quinpirole or muscimol or muscimol + quinpirole.

spine formation and GABAA receptors, and thus negatively regulate D2R-induced spine formation through ERK signaling. DA receptors are present throughout the soma and dendrites of the neuron, but accumulating evidence indicates that they are concentrated in dendritic spines [25]. It has been reported that dendritic spine formation in cultured striatal medium spiny neurons co-cultured with DA neurons is facilitated by DA through

DA receptors, including D2R [26]. However, the exact mechanism underlying this regulation is not well understood and assessment of DA signaling in spines is essentially still lacking. In this context, D2R-mediated ERK activation could be a prominent signaling pathway through which DA exerts an important role in spine formation. This indicates also that D2Rs can control the excitability and synaptic properties of spines which can further modulate

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the DA-associated signaling in the hippocampal synaptic environment. Therefore, our present data provide the potential basis of D2R-mediated excitability in hippocampal networks, through D2Rinduced ERK activation. We also cannot rule out the possibility that D2R-interacting proteins can exert a role in spine formation. Recent studies revealed a direct interaction between D2Rs and several scaffolding, cytoskeletal, and receptor proteins in the postsynaptic density (PSD) such as the actin binding protein (ABP-280; filamin A) [27], or spinophilin, an F-actin-associating postsynaptic protein in the PSD [28], which may provide a platform that links D2Rs to the actin cytoskeleton and downstream signaling in dendritic spines. It is well accepted that schizophrenia is associated with abnormally enhanced activity within the DA system [29,30]. Further, it has been reported that enhanced hippocampal activity correlates with enhanced mesolimbic DA neuron activity in schizophrenic patients suggesting that this hyperactivity within hippocampal subfields observed in schizophrenia is attributable to a decrease in GABAergic signaling [30,31]. This decreased GABAergic signaling results in subsequent hyperactive hippocampal activity, which in turn drives a pathologically enhanced DA system. This indicates that modulation of GABAergic signaling could be important, as well as that of DA signaling, in the pathology of schizophrenia. Our findings here, thus, lead us to speculate that negative regulation of GABAA receptors on D2R-mediated spine formation through ERK signaling, which can enhance the excitability of hippocampal neurons, could be critical in ensuring homeostatic neuronal activity in the synaptic environment of the hippocampus. In our experiment, hippocampal neuronal cells and mesencephalic neuronal cells showed different response to GABAA receptor ligands on D2R-induced ERK activation. This might be related with the differential regional expression of GABAA receptor in embryonic periods. It has been reported that in late- embryonic stage, the expression of GABAA receptor subunit mRNAs are very low or undetectable in mesencephalon [32], while in hippocampal neurons GABAA receptor subunit mRNAs are expressed significantly [19,32]. This indicates that our D2R-induced ERK activation is specific with the expression of GABAA receptor in hippocampal neurons but this regulation is absent in mesencephalon due to the lack of GABAA receptor expression. In this context, the observed effect of pretreatment of bicuculline on D2R-mediated ERK activation in mesencephalic neurons may be independent of GABAA receptor. It has been reported that GABAA receptors exert negative regulation on ERK activation in the hippocampus in association with spatial learning and memory [33,34], memory retrieval [35], and object recognition memory [15]. Therefore, it will be interesting to see whether these regulations include D2R-GABAA receptor interactions and how this receptor cross-talk can be translated into behavioral modulation. In conclusion, the findings of this study provide us with a novel function for D2Rs in association with GABAA receptors through the regulation of ERK signaling on regulation of the hippocampal synaptic plasticity. Further studies should attempt to determine whether this signaling may play a role in hippocampal D2R function in the control of related behavioral functions such as the detection of novel information and modulation of memory formation and retrieval.

Acknowledgments This work was supported by Mid-Career Researcher Program from National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIP) (Grant no. 2014R1A2A2A01003337) and by a Korea University Grant.

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