Dopamine D4 Receptors Regulate GABAA Receptor Trafficking via an Actin/Cofilin/Myosin-dependent Mechanism*

The GABAA receptor-mediated inhibitory transmission in prefrontal cortex (PFC) is implicated in cognitive processes such as working memory. Our previous study has found that GABAAR current is subject to the regulation of dopamine D4 receptors, a PFC-enriched neuromodulator critically involved in various mental disorders associated with PFC dysfunction. In this study, we have investigated the cellular mechanism underlying D4 modulation of GABAARs. We found that the density of surface clusters of GABAAR β2/3 subunits was reduced by D4, suggesting that the D4 reduction of GABAAR current is associated with a decrease in functional GABAARs at the plasma membrane. Moreover, the D4 reduction of GABAAR current was blocked by the actin stabilizer phalloidin and was occluded by the actin destabilizer latrunculin, suggesting that D4 regulates GABAAR trafficking via an actin-dependent mechanism. Cofilin, a major actin depolymerizing factor whose activity is strongly increased by dephosphorylation at Ser3, provides the possible link between D4 signaling and the actin dynamics. Because myosin motor proteins are important for the transport of vesicles along actin filaments, we also tested the potential involvement of myosin in D4 regulation of GABAAR trafficking. We found that dialysis with a myosin peptide, which competes with endogenous myosin proteins for actin-binding sites, prevented the D4 reduction of GABAAR current. These results suggest that D4 receptor activation increases cofilin activity presumably via its dephosphorylation, resulting in actin depolymerization, thus causing a decrease in the myosin-based transport of GABAAR clusters to the surface.

The GABA A receptor-mediated inhibitory transmission in prefrontal cortex (PFC) is implicated in cognitive processes such as working memory. Our previous study has found that GABA A R current is subject to the regulation of dopamine D 4 receptors, a PFC-enriched neuromodulator critically involved in various mental disorders associated with PFC dysfunction. In this study, we have investigated the cellular mechanism underlying D 4 modulation of GABA A Rs. We found that the density of surface clusters of GABA A R ␤2/3 subunits was reduced by D 4 , suggesting that the D 4 reduction of GABA A R current is associated with a decrease in functional GABA A Rs at the plasma membrane. Moreover, the D 4 reduction of GABA A R current was blocked by the actin stabilizer phalloidin and was occluded by the actin destabilizer latrunculin, suggesting that D 4 regulates GABA A R trafficking via an actin-dependent mechanism. Cofilin, a major actin depolymerizing factor whose activity is strongly increased by dephosphorylation at Ser 3 , provides the possible link between D 4 signaling and the actin dynamics. Because myosin motor proteins are important for the transport of vesicles along actin filaments, we also tested the potential involvement of myosin in D 4 regulation of GABA A R trafficking. We found that dialysis with a myosin peptide, which competes with endogenous myosin proteins for actin-binding sites, prevented the D 4 reduction of GABA A R current. These results suggest that D 4 receptor activation increases cofilin activity presumably via its dephosphorylation, resulting in actin depolymerization, thus causing a decrease in the myosin-based transport of GABA A R clusters to the surface.
Prefrontal cortex (PFC), 2 a brain region strongly associated with cognitive and emotional processes (1), is particularly critical for working memory, a mechanism for encoding and maintaining newly acquired, task-relevant information (2). Working memory relies on the coordinated sustained firing of PFC pyramidal neurons between the temporary presentation of a stimulus cue and the later initiation of a behavioral response (2). The synchronization of pyramidal neuron activity during working memory processes is controlled by GABAergic interneurons (3,4). Impairments in GABA-mediated inhibition in the PFC have been considered a major mechanism for working memory disturbances in schizophrenia (5).
GABAergic neurotransmission is mediated by GABA A receptors, the heteropentameric ligand-gated ion channels located at inhibitory synapses at soma and proximal dendrites (6). After being assembled in endoplasmic reticulum, the GABA A R complex is targeted and clustered at synapses by receptor-associated proteins via unclear mechanisms (7). Postsynaptic GABA A Rs undergo constitutive endocytosis via a clathrin-mediated dynamin-dependent pathway (8). Depending on the subunit composition, GABA A Rs are internalized to peripheral endosomal compartments or perinuclear late endosomes (9,10). Alterations in the assembly, trafficking, or function of GABA A Rs can lead to changes in GABAergic inhibition, which is often linked to the pathophysiology of various neurological disorders (11). For example, the GABA A R ␣ 2 subunit in the axon initial segment of PFC pyramidal neurons is up-regulated in schizophrenia (12). Schizophrenic patients show altered ratios of alternatively spliced transcripts of GABA A R ␥2 subunit in PFC (13). Decreased GABA A R clustering results in enhanced anxiety (14).
PFC is a major target of dopaminergic input from the ventral tegmental area (15,16), and dopamine plays a key role in regulating PFC functions such as working memory (17,18). Evidence suggests that the dopamine D 4 receptor, which is highly enriched in PFC (19,20), is critically involved in neuropsychiatric disorders associated with PFC dysfunction. A D 4 gene polymorphism that weakens D 4 receptor function is strongly linked to attention deficit-hyperactivity disorder (21,22). Elevated D 4 receptor expression has been demonstrated in schizophrenic patients (23), and D 4 receptors have high affinity for atypical antipsychotic drugs (24,25). D 4 receptor antagonists can alleviate cognitive deficits induced by stress (26) or long term treatment with the psychotomimetic drug phencyclidine (27,28). D 4 receptor-deficient mice show reduced novelty seeking and cortical hyperexcitability (29,30).
To understand the mechanism of D 4 actions in PFC, it is important to identify its cellular targets key to PFC functions such as working memory. One of our previous studies has demonstrated that GABA A receptors are subject to D 4 regulation in PFC pyramidal neurons (31). In this study, we have revealed the mechanism underlying this regulation.

EXPERIMENTAL PROCEDURES
Acute Dissociation Procedure and Primary Culture Preparation-PFC neurons from young adult (3-4 weeks postnatal) rats were acutely dissociated using procedures described previously (32,33). All of the experiments were carried out with the approval of State University of New York at Buffalo Animal Care Committee. After incubation of brain slices in NaHCO 3 -buffered saline, PFC was dissected and placed in an oxygenated chamber containing papain (0.8 mg/ml; Sigma) in HEPES-buffered Hanks' balanced salt solution (Sigma) at room temperature. After 35 min of enzyme digestion, the tissue was rinsed three times with a low Ca 2ϩ saline and mechanically dissociated with a graded series of firepolished Pasteur pipettes. The cell suspension was then plated into a 35-mm Lux Petri dish, which was then placed on the stage of a Zeiss Axiovert S100 inverted microscope.
Rat PFC cultures were prepared as previously described (34). Briefly, PFC was dissected from 18-day rat embryos, and the cells were dissociated by incubating with 0.25% trypsin for 30 min and subsequent trituration through a Pasteur pipette cells. The cells were plated on coverslips (coated with poly-L-lysine) in Dulbecco's modified Eagle's medium with 10% fetal calf serum at a density of 0.75 ϫ 10 5 cells/cm 2 . When neurons attached to the coverslip within 4 h, the medium was changed to Neurobasal with B27 supplement. The neurons were maintained for 2-3 weeks before being used.
Whole Recordings were obtained using an Axopatch 200B amplifier that is controlled and monitored with a computer running pClamp 8 with a DigiData 1320 series interface. Electrode resistances were typically 2-4 M⍀ in the bath. After seal rupture, series resistance (4 -10 M⍀) was compensated (70 -90%) and periodically monitored. The cell membrane potential was held at 0 mV. GABA (50 M) was applied for 2 s every 30 s to minimize desensitization-induced decrease of current amplitude. Drugs were applied using a gravity-fed "sewer pipe" system. The array of application capillaries (ϳ150-m inner diameter) was positioned a few hundred micrometers from the cell being recorded. Solution changes were affected by the SF-77B faststep solution stimulus delivery device (Warner Instruments, Hamden, CT).
Data analyses were performed with Clampfit (Axon Instruments, Sunnyvale, CA) and Kaleidagraph (Albeck Software, Reading, PA). For analysis of statistical significance, ANOVA tests were performed to compare the differential degrees of current modulation between groups subjected to different treatments.
Recording of evoked IPSC in PFC slices used the same internal solution as what was used for mIPSC recording in cultures. The slice (300 m) was placed in a perfusion chamber attached to the fixed-stage of an upright microscope (Olympus) and submerged in continuously flowing oxygenated artificial cerebral spinal fluid containing D-AP5 (20 M) and DNQX (20 M). The cells were visualized with a 40ϫ water immersion lens and illuminated with near infrared light, and the image was detected with an infrared-sensitive CCD camera (Olympus, Center Valley, PA). A Multiclamp 700A amplifier was used for slice recordings (Axon Instruments). Tight seals (2-10 G⍀) from visualized pyramidal neurons were obtained by applying negative pressure. The membrane was disrupted with additional suction and the whole cell configuration was obtained. The access resistances ranged from 13 to 18 M⍀ and were compensated 50 -70%. The cells were held at Ϫ70 mV. Clampfit (Axon Instruments) was used to analyze evoked synaptic activity.
The agents used such as N-(methyl)-4-(2-cyanophenyl)piperazinyl-3-methybenzamide maleate (PD168077; Tocris, Ballwin, MO), colchicine, phalloidin, latrunculin B (Calbiochem, San Diego, CA), dynamin inhibitory peptide (Tocris, Ballwin, MO), p-cofilin peptide, cofilin peptide, and a scrambled peptide were made up as concentrated stocks in water or Me 2 SO and stored at Ϫ20°C. The final Me 2 SO concentration in all applied solutions was Ͻ0.1%. No change on GABA A R currents has been observed with this concentration of Me 2 SO. Stocks were thawed and diluted immediately before use. The amino acid sequence for the myosin peptide is KLFNDPNIGKKGARGKKGKKGRAQKGAN.
Immunocytochemical Staining-After treatment, the cultures were fixed in 4% paraformaldehyde for 20 min and incubated in 5% bovine serum for 1 h. For GABA A R surface expression, cultured neurons (nonpermeabilized) were incubated with an antibody against GABA A R ␤2/3 extracellular region (1:50; Chemicon, Billerica, MA) for 2 h at room temperature. After washing, the neurons were permeabilized with 0.1% Tri-ton for 10 min and then incubated with MAP2 antibody (1:500; Santa Cruz, Santa Cruz, CA) for 2 h at room temperature. Following washing, the neurons were incubated with Alexa 488conjugated secondary antibody (1:200; Invitrogen) and Alexa 594-conjugated secondary antibody (1:500; Invitrogen) for 1 h at room temperature. After washing, the coverslips were mounted on slides with VECTASHIELD mounting media (Vector Laboratories, Burlingame, CA).
Fluorescent images were obtained using a 100ϫ objective with a cooled charge-coupled device camera mounted on a Nikon microscope. All of the specimens were imaged under identical conditions and analyzed with identical parameters using ImageJ software. Control and PD168077treated neurons with similar MAP2 staining were selected for analysis. To define dendritic clusters, a single threshold was chosen manually, so that clusters corresponded to puncta of at least 1.5-fold intensity of the diffuse fluorescence on the dendritic shaft. Three to four independent experiments for each of the treatments were performed. On each coverslip, the cluster density, cluster size, and cluster fluorescence intensity of several neurons (two or three dendritic segments of at least 20 m in length/neuron) were measured. Quantitative analyses were conducted blindly (without knowledge of experimental treatment).
Western Blots-After treatment, equal amounts of protein from culture homogenates were separated on 7.5% acrylamide gels and transferred to nitrocellulose membranes. The blots were blocked with 5% nonfat dry milk for 1 h at room temperature and then were incubated with the anti-p-cofilin (1:250; Cell Signaling, Danvers, MA), anti-cofilin (1:250; Cell Signaling), or anti-actin (1:500; Cell Signaling) for 3 h at room temperature. After washing, the blots were incubated with the horseradish peroxidase-conjugated antirabbit antibody (1:1000; Amersham Biosciences) for 2 h at room temperature. After washing, the blots were exposed to the enhanced chemiluminescence substrate. Quantification was obtained from densitometric measurements of immunoreactive bands on films using National Institutes of Health Image software.  application evoked a partially desensitizing outward current in neurons (held at 0 mV) that could be completely blocked by the GABA A R antagonist bicuculline (30 M; data not shown). As shown in Fig. 1A, application of PD168077 (30 M) caused a reversible reduction of GABA A R current amplitudes in dissociated PFC pyramidal neurons (16.8 Ϯ 1.7%, n ϭ 15). Consistent with our previous findings (31), this effect of PD168077 was blocked by the specific D 4 antagonist L-74172 (10 M, data not shown), suggesting the mediation by D 4 receptors.

Activation of D 4 Receptors Reduces GABA A R Channel Currents and Surface Expression in PFC Pyramidal
To examine the impact of D 4 receptors on GABAergic synaptic transmission, we further measured IPSC evoked by electrical stimulation of synaptic GABA A receptors. As shown in Fig. 1B, bath application of PD168077 (40 M) to PFC slices caused a reversible reduction of IPSC amplitudes (34.6 Ϯ 2.6%, n ϭ 7), whereas IPSC amplitudes remained stable in control neurons when no PD168077 was applied. Moreover, we measured miniature IPSC, a response from quantal release of single GABA vesicles. As shown in Fig. 1C, PD168077 (30 M) caused a reversible reduction of mIPSC amplitudes in cultured PFC pyramidal neurons (17.8 Ϯ 3.5%, n ϭ 23). Taken together, these results suggest that D 4 receptors down-regulate GABA A R function at the synapse.
Next, we tested whether the D 4 -induced down-regulation of GABA A R function was due to a decrease in GABA A R surface expression. We labeled surface GABA A receptors using an antibody that targets the extracellular region of GABA A R ␤2/3 subunit in PFC cultures. Neurons were co-stained with MAP2, a dendritic marker. As illustrated in Fig. 1D, surface GABA A Rs were clustered around the soma and proximal dendrites. In cells treated with PD168077 (30 M, 10 min), GABA A R surface clusters were substantially reduced. Quantification of immunocytochemical images (Fig. 1E) indicates that the density of GABA A R surface clusters (number of clusters/20 m dendrite) was significantly reduced by PD168077 (control: 13.9 Ϯ 1.4, n ϭ 12; PD168077: 9.4 Ϯ 1.4, n ϭ 12, p Ͻ 0.05, ANOVA). PD168077 did not cause a significant change in the size of GABA A R surface clusters or the fluorescence intensity (normalized to MAP2 immunofluorescence) of GABA A R surface clusters. These results suggest that D 4 receptor activation leads to a decrease of GABA A R surface cluster density, which is associated with the D 4 -induced reduction of whole cell GABA A R current, evoked IPSC, and miniature IPSC amplitude.
The Actin Cytoskeleton Is Involved in D 4 Regulation of GABA A R Currents-Next, we examined the underlying mechanism for D 4 reduction of GABA A Rs at the cell surface. Previous studies have shown that GABA A receptors are removed from the plasma membrane mainly by clathrin/dynamin-mediated endocytosis (36,37). To test whether D 4 receptor activation induces GABA A R endocytosis, we dialyzed neurons with a dynamin inhibitory peptide, which competitively blocks dynamin from binding to amphiphysin, thus preventing endocytosis (38). The effectiveness of this peptide to block GABA A R endocytosis has been demonstrated in our previous studies (39). As shown in Fig. 2 (A and B), PD168077 reduced GABA A R current in the presence of dynamin inhibitory peptide (50 M, 15.6 Ϯ 2.5%, n ϭ 6), which was similar to the effect of PD168077 in the absence of this peptide (16. 8 Ϯ 1.7%, n ϭ 6). These results suggest that D 4 reduction of GABA A R current is not through increased endocytosis of GABA A Rs.
Previous studies have suggested the involvement of cytoskeleton proteins in regulating GABA A receptor current and surface stability (40,41); thus we investigated the potential role of microtubules and/or actin in D 4 regulation of GABA A R current. As shown in Fig. 2C, dialysis with the actin stabilizing compound phalloidin (12.5 M) largely blocked the capability of PD168077 to reduce GABA A R current. Phalloidin itself had little effect on basal GABA A R current (5.1 Ϯ 1.0%, n ϭ 5). As summarized in Fig. 2D, the effect of PD168077 was significantly (p Ͻ 0.005, ANOVA) smaller in phalloidin-loaded neurons (6.1 Ϯ 0.5%, n ϭ 4), compared with control neurons (16.1 Ϯ 1.2%, n ϭ 6). Conversely, application of latrunculin B, an actin depolymerizing compound, caused a decline of GABA A R current (31.1 Ϯ 2.0%, n ϭ 7) and largely occluded the effect of subsequently applied PD168077 (Fig. 2E). However, the microtubule destabilizing compound colchicine, which reduced basal GABA A R current (27.2 Ϯ 3.0%, n ϭ 8), failed to alter the reducing effect of PD168077 (Fig. 2E). As summarized in Fig. 2F, neurons dialyzed with latrunculin B showed a significantly (p Ͻ 0.005, ANOVA) smaller effect of PD168077 (6.1 Ϯ 1.1%, n ϭ 6), compared with control neurons (16.1 Ϯ 1.2%, n ϭ 6) or neurons perfused with colchicine (14.1 Ϯ 1.2%, n ϭ 14). Consistently, bath application of latrunculin B also occluded the effect of PD168077 on evoked IPSC in PFC slice recordings (Fig. 2G, 6.2 Ϯ 2.0%, n ϭ 5). These results suggest that D 4 reduces GABA A R current via an actin-dependent mechanism. D 4 Reduction of GABA A R Current Is Dependent upon the Actin Depolymerizing Factor Cofilin-Next, we investigated the link between D 4 receptor signaling and actin cytoskeleton. The dynamics of actin assembly is regulated by cofilin, a major actin depolymerizing factor (42). The actin depolymerizing activity of cofilin is greatly increased by dephosphorylation at Ser 3 (43,44). In vitro studies have shown that protein phosphatase 1 (PP1) can lead to the dephosphorylation and activation of cofilin (45). Our previous study has found that D 4 regulation of GABA A R current depends on activation of the anchored PP1 (31). Thus, we speculated that D 4 activation might induce actin depolymerization by dephosphorylating cofilin via PP1, thus leading to the reduced GABA A R synaptic trafficking along actin cytoskeleton. To test this, we first examined the impact of D 4 on cofilin activity using a Ser 3 phospho-cofilin antibody in cultured PFC neurons. As shown in Fig. 3A, application of PD168077 (30 M, 10 min) significantly reduced the level of Ser 3 -phosphorylated (inactive) cofilin (65.1 Ϯ 3.1% of control, n ϭ 5; p Ͻ 0.005, ANOVA), and this effect was blocked by pretreatment with the PP1 inhibitor okadaic acid (1 M, 40 min, 95.3 Ϯ 3.1% of control, n ϭ 3; p Ͼ 0.05, ANOVA). The level of total cofilin or actin was not changed. These results suggest that D 4 activation leads to the dephosphorylation and activation of cofilin through a PP1-dependent mechanism.
The Actin Motor Protein, Myosin, Is Involved in D 4 Regulation of GABA A R Current-Given the actin dependence of D 4 regulation of GABA A R current, we further examined the potential involvement of actin-based motor proteins. Myosin, a family of motor proteins that move on F-actin, has been found to be critical for the trafficking of AMPARs (48 -50); however, its involvement in GABA A R trafficking is unknown. Thus, we dialyzed neurons with a synthetic peptide derived from the conserved actinbinding site of myosin proteins, which competes with endogenous myosin for actin binding and therefore impairs myosin-based trafficking along actin filaments (51). As shown in Figs. 4 (A and B), in the presence of the myosin peptide, the effect of D 4 on GABA A R current in dissociated PFC neurons was significantly (p Ͻ 0.005, ANOVA) smaller (7.1 Ϯ 0.8%, n ϭ 14) compared with control conditions (14.6 Ϯ 1.0%, n ϭ 14).
Next, we examined the impact of the myosin peptide on D 4 modulation of GABAergic transmission. As shown in Fig. 4C, bath application of PD168077 to PFC cultures caused a significant (p Ͻ 0.001; Kolmogorov-Smirnov test) reduction of mIPSC amplitudes, as indicated by a leftward shift on the mIPSC distribution; however, this effect was prevented by the myosin peptide. As summarized in Fig. 4D, the effect of D 4 on mIPSC amplitude was significantly (p Ͻ 0.005, ANOVA) reduced in neurons dialyzed with the myosin peptide (6.6 Ϯ 2.9%, n ϭ 6), compared with control conditions (19.8 Ϯ 2.2%, n ϭ 7). Similarly, the myosin peptide, but not a scrambled control peptide, significantly (p Ͻ 0.005, ANOVA) blocked the reducing effect of D 4 on evoked IPSC in PFC slices (Fig. 4, E and F, with myosin peptide, 7.8 Ϯ 1.8%, n ϭ 8; with scrambled peptide, 37.1 Ϯ 5.3%, n ϭ 8). These data suggest that D 4 affects myosin-mediated transport of GABA A Rs along actin filaments.

DISCUSSION
In this study, we have revealed that D 4 receptor activation in PFC pyramidal neurons reduces GABA A R-mediated channel current and inhibitory transmission via a mechanism involving actin-based trafficking of GABA A Rs to the synaptic membrane. Our results suggest that D 4 triggers the PP1-mediated dephosphorylation and activation of cofilin, the major actin depolymerizing factor, leading to the loss of actin stability. Consequently, the myosin motor-mediated transport of GABA A R-containing vesicles along F-actin is interrupted, resulting in reduced GABA responses.
The trafficking of functional GABA A Rs is fundamental for establishing and maintaining inhibitory transmission (52). There is evidence suggesting that newly assembled GABA A Rs are delivered to extrasynaptic sites and then rapidly imported to synaptic sites through lateral diffusion (53). Surface GABA A Rs are constitutively endocytosed from the cell surface via a dynamin/clathrin-dependent mechanism that is regulated by phosphorylation (8,54). Internalized GABA A Rs are either rapidly recycled back to the cell surface or targeted for lysosomal degradation, and this sorting decision is regulated by a direct interaction of GABA A Rs with Huntingtin-associated protein 1 (55).
Using acutely dissociated neurons, primary cultures and brain slices, our electrophysiological data show that D 4 recep-tor activation reduces functional GABA A Rs at both synaptic and extrasynaptic sites, which is consistent with the D 4 -induced reduction of surface GABA A R clusters on soma and processes illustrated by immunocytochemical results. The pharmacological experiments with agents disturbing actin dynamics suggest that D 4 down-regulates GABA A R trafficking and function by reducing actin stability. In agreement with this, it has been shown that actin depolymerization can lead to a decrease in GABA A R clusters at the cell surface (40).
Several studies have demonstrated the role of actin cytoskeleton in regulating AMPA-type glutamate receptor cluster distribution (56), surface expression (57), and channel internalization (58,59). However, the involvement of actin in anchoring and clustering GABA A Rs at inhibitory synapses is much less clear. The actin-binding protein radixin has been identified as the first directly interacting molecule that anchors GABA A Rs at cytoskeletal elements (41). Depletion of radixin expression or replacement of the radixin/F-actin binding motif interferes with GABA A R ␣ 5 cluster formation (41). Although radixin only associates with GABA A R ␣ 5 subunit, which mainly localizes at extrasynaptic sites and mediates tonic inhibition, other GABA A R subunits might be targeted to synapses via actin filaments by interacting with other actin-associated scaffolding proteins. It is possible that the D 4 -induced actin depolymerization disrupts the interaction of GABA A Rs with their anchoring proteins, leading to the loss of GABA A Rs at the synapse.
further demonstrated that the D 4 regulation of GABA A R-mediated ionic current and inhibitory transmission requires the activation of cofilin.
Because D 4 increases the actin-depolymerizing activity of cofilin, we would expect to see changes in F-actin organization by D 4 activation. Indeed, PD168077 treatment led to a marked loss of F-actin clusters and a diffuse labeling pattern of F-actin in cultured PFC neurons (data not shown). These results suggest that D 4 activation can alter actin dynamics, thus leading to changes in actin-based trafficking of receptors.
Myosin proteins are actin-associated motors whose major function is to control the transport of organelles along the actin filament (62). About 40 myosin genes (grouped into 12 distinct classes) have been identified. These motors are composed of a conserved N-terminal motor domain followed by a coiled-coil region and a globular C-terminal tail containing the cargo binding domain (63). Using a peptide against the actinbinding region that is conserved for most myosin proteins (51), we have demonstrated the role of myosin motor proteins in D 4 regulation of GABA A R trafficking and function. Class V of myosins, which is thought to regulate the trafficking of organelles and associated proteins in neurons (62), has been implicated in AMPAR trafficking (48,50). However, we found that blocking myosin V function with a specific antibody did not affect the D 4 regulation of GABA A R current (supplemental Fig. S1, A and B), suggesting the lack of involvement of myosin V in GABA A R trafficking. In agreement with this, it has been shown that GABA A R-mediated IPSC is unaffected in neurons transfected with dominant-negative myosin V (50). Furthermore, we tested the effect of blebbistatin, a myosin II inhibitor (64), on D 4 regulation of GABA A Rs. As shown in supplemental Fig. S1 (C-E), blebbistatin (2.5 M) failed to alter the reducing effect of PD168077 on mIPSC amplitude (control, 19.8 Ϯ 2.2%, n ϭ 7; with blebbistatin, 19.1 Ϯ 0.4%, n ϭ 5), suggesting a lack of involvement of myosin II. It awaits to be identified which subtype of myosin proteins is involved in the regulation of actin-based GABA A R trafficking.
In the central nervous system, dopamine, by activating different receptors, regulates GABA A receptors via distinct mechanisms. D 1 receptor has been shown to reduce GABA A R currents in neostriatum by activating a PKA/DARPP-32/PP1 signaling cascade to increase GABA A R ␤1 subunit phosphorylation (65). D 3 receptor has been shown to suppress postsynaptic GABA A R currents in nucleus accumbens by increasing the phospho-dependent endocytosis of GABA A receptors (39). The present study has revealed that D 4 receptors regulate GABA A R trafficking and function via an actin/cofilin/myosindependent mechanism in prefrontal cortex. These studies provide a framework for understanding the role of dopamine receptors in regulating the efficacy of GABA A R-mediated inhibitory synaptic transmission of diverse brain regions.