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Cerebral Cortex Advance Access originally published online on December 7, 2005
Cerebral Cortex 2006 16(10):1377-1388; doi:10.1093/cercor/bhj084
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Feature Article

Ambient GABA Promotes Cortical Entry of Tangentially Migrating Cells Derived from the Medial Ganglionic Eminence

Verginia C. Cuzon1,4, Pamela W. Yeh1,4, Qing Cheng2 and Hermes H. Yeh1,3,4

1 Center for Aging and Developmental Biology, University of Rochester Medical Center, Rochester, NY 14642, USA, 2 Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA and 3 Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA, 4 Current address: Department of Physiology, Dartmouth Medical School, Dartmouth Hitchcock Medical Center, Lebanon, NH 03756, USA

Address correspondence to Dr Hermes H. Yeh, Department of Physiology, Dartmouth Medical School, Lebanon, NH 03756, USA. Email: Hermes.Yeh{at}Dartmouth.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During corticogenesis, cells from the medial ganglionic eminence (MGE) migrate tangentially into the neocortical anlage. Here we report that {gamma}-aminobutyric acid (GABA), via GABAA receptors, regulates tangential migration. In embryonic telencephalic slices, bicuculline produced an outward current in migrating MGE-derived cells in the neocortex, suggesting the presence of and tonic activation by ambient GABA. Ambient GABA was also present in the MGE, although this required demonstration using as bioassay HEK293 cells expressing high-affinity {alpha}6/ß2/{gamma}2s recombinant GABAA receptors. The concentration of ambient GABA was 0.5 ± 0.1 µM in both regions. MGE-derived cells before the corticostriate juncture (CSJ) were less responsive to GABA than those in the neocortex, and profiling of GABAA receptor subunit transcripts revealed different expression patterns in the MGE vis-à-vis the neocortex. These findings suggest a dynamic expression of GABAA receptor number or isoform as MGE-derived cells enter the neocortex and become tonically influenced by ambient GABA. Treatment with bicuculline or antibody against GABA did not affect migration of MGE-derived cells before the CSJ but decreased "crossing index," reflecting impeded migration past the CSJ into the neocortex. Treatment with diazepam or addition of exogenous GABA increased crossing index. We conclude that ambient GABA promotes cortical entry of tangentially migrating MGE-derived cells.

Key Words: corticogenesis • developing neocortex • GABAA receptor • GFP-MGE • telencephalic slice coculture


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Migration during corticogenesis plays an important role in orchestrating the assembly of cortical neurons within the laminated layout of the neocortex. Errors in the migration process, such as number, timing, and regionalization of neocortical neurons, have been implicated in disorders such as epilepsy, schizophrenia, and autism (reviewed in Gleeson and Walsh 2000Go; Monuki and Walsh 2001Go; Ross and Walsh 2001Go; Kato and Dobyns 2003Go; Mochida and Walsh 2004Go). Radially and tangentially migrating cells in the developing neocortex arise from different progenitors (Mione and others 1997Go; Tan and others 1998Go). The pyramidal cells of the neocortex arise from progenitors in the ventricular zone and migrate along radial glia to their respective cortical layers (Rakic 1972Go). In addition, there is abundant evidence that a vast majority of GABAergic cortical interneurons proliferate and tangentially migrate from the ganglionic eminence (GE) to the neocortex (DeCarlos and others 1996Go; Anderson and others 1997Go, 1999Go, 2001Go; Tamamaki and others 1997Go; Lavdas and others 1999Go; Wichterle and others 2001Go; Nery and others 2002Go; Rakic and Zecevic 2003Go; López-Bendito and others 2004Go; Xu and others 2004Go). The medial ganglionic eminence (MGE) provides the initial and predominant wave of tangentially migrating neurons, whereas the lateral ganglionic eminence (LGE) and caudal GE contribute a subsequent smaller contingent. Factors that regulate the tangential migration of GE cells into the neocortex remain to be elucidated.

In the present study, we tested the hypothesis that GABA mediates the tangential migration of MGE-derived cells into the neocortex. GABA is in a favorable position to play such a role (e.g., López-Bendito and others 2003Go; Manent and others 2005Go; reviewed by Nguyen and others 2001Go; Varju and others 2001Go; Represa and Ben-Ari 2005Go). Specifically, early in corticogenesis, GABA has been shown to be present in the developing neocortex along the major migratory routes of GE cells, namely, the subventricular zone (SVZ), intermediate zone (IZ), and marginal zone (MZ) (Van Eden and others 1989Go; Tamamaki and others 1997Go; Lavdas and others 1999Go; Anderson and others 2001Go; Jimenez and others 2002Go; Polleux and others 2002Go). Immature neurons in the developing cortex express GABAA receptor subunit mRNAs and functional receptors (Araki and others 1992Go; Laurie and others 1992Go; Poulter and others 1992Go; LoTurco and others 1995Go; Owens and others 1996Go, 1999Go). Here, we established an isochronic slice culture system in which cells migrating out of explants of MGE taken from green fluorescent protein (GFP)–expressing mouse embryos could be monitored continuously when placed onto the MGE in slices derived from wild-type littermates. Patch clamp electrophysiological experiments demonstrated an ambient level of GABA in the developing neocortex that is present at an estimated concentration of 0.5 µM. Reverse transcription–polymerase chain reaction (RT-PCR)–based expression revealed different cadres of GABAA receptor subunit transcripts in the MGE and neocortex. MGE-derived cells display increased responsiveness to GABA as migration progresses from the MGE, past the corticostriate juncture (CSJ), and into the neocortex. Importantly, GABA, via GABAA receptors, promotes the entry of MGE-derived cells into the neocortex. These findings, in toto, implicate GABA as a developmental neurotransmitter that regulates tangential migration of neocortex-bound MGE cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of Embryonic Telencephalic Slice Cocultures

All animal procedures were approved by the University of Rochester Medical Center Animal Resource Review Committee. Transgenic mice expressing GFP under the control of a ß-actin/cytomegolovirus promoter/enhancer sequence were mated with wild-type C57-BL/6J mice of the opposite sex (Okabe and others 1997Go). Time-pregnant dams at embryonic days E13.5–E15.5 were asphyxiated using CO2, the embryos were removed by cesarean section, and the GFP-expressing embryos were identified using UV fluorescence (Zelco, Mt Vernon, New York). The brains were quickly dissected and immersed in ice-cold oxygenated slicing medium consisting of Dulbecco's Modified Eagles' Medium (DMEM)/F12 (1:1; GIBCO, Grand Island, New York) supplemented with extra glucose (6 mg/ml) and penicillin/streptomycin (Cellgro, Herndon, Virginia). The brains were then embedded in 3.5% low–melting point agarose (Invitrogen, Carlsbad, California) prepared using the slicing medium, and 250-µm coronal slices from the anterior half of the cerebral hemisphere were obtained using vibroslicer (WPI, Sarasota, Florida). To obtain isochronic cocultures derived from green fluorescent protein–expressing medial ganglionic eminence (GFP-MGE) explants and wild-type embryonic telencephalic slices, the MGE was microdissected from GFP-expressing slices and briefly incubated in 0.3% methyl green. The GFP-MGE explants were then minced into pieces of relatively uniform size (approximately 150 x 150 µm), aspirated into a glass pipette, and transferred onto the MGE of slices obtained from wild-type littermates. Only wild-type slices in which the MGE and LGE are demarcated by the ganglionic sulcus and clearly distinguishable were used as host for the placement of GFP-MGE explants. Brief incubation in methyl green facilitated visualization of the GFP-MGE explants during placement (Fig. 1A1,A2). When labeling cells with the cell tracker 4-chloromethyl benzoyl amino tetramethyl rhodamine (CMTMR), a small amount of tungsten dust particles (0.4 µm; BioRad, Hercules, California) coated with CMTMR (1 mM diluted in ethylene dichloride; Molecular Probes, Eugene, California) was deposited onto wild-type MGE with the aid of a glass pipette, following the procedure of Alifragis and others (2002)Go. Cells were visualized under fluorescence illumination.


Figure 1
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  		Figure 1. GFP-MGE–derived cells comigrate with endogenous cells. (A) A GFP-MGE explant incubated briefly in 0.3% methyl green is seen placed on a wild-type E14.5 telencephalic slice under bright field (A1) and epifluorescence (A2). Scale bar = 200 µm. (B) Fluorescence image of GFP-MGE cells migrating into the cortex from the explant. Scale bar = 200 µm. (C) GFP-MGE cells comigrating with endogenous MGE-derived cells labeled with CMTMR. Arrows indicate examples of migrating GFP-MGE cells. Arrowheads indicate examples of endogenous migrating cells labeled with CMTMR placed in the MGE. Scale bar = 50 µm. (D1–5) Sequential time-lapse images of a GFP-MGE cell in the IZ of the neocortex. Scale bar = 10 µm. Such images were used to calculate the migration rate of GFP-MGE and CMTMR-labeled cells (E), expressed as mean ± standard error. Data in (E) were derived from 9 slices, with 9 cells analyzed per slice.

 
Embryonic slice cocultures were maintained in culture medium containing DMEM/F12 (1:1) supplemented with glucose, 10% fetal calf serum (Hyclone, Logan, Utah), L-glutamine (2 mM; Sigma, St Louis, Missouri), and penicillin/streptomycin. The slices were placed on 1 x 1–cm pieces of Spectra/Mesh nylon (10-µm open mesh; Spectrum Labs, Rancho Dominguez, California) that were supported by a 100-µm-diameter platinum insert to maximize access to the culture medium and to achieve an air–medium interface culture condition. The slice cocultures were maintained for 1–2 days in a humidified 5% CO2 atmosphere at 37 °C. The following final concentrations of agents that affect GABAA receptor function (all purchased from Sigma unless stated) were added to the slice cultures: 0.5 µM diazepam, 20 µM bicuculline, 1 µM GABA, a polyclonal antibody against GABA (rabbit anti-GABA, 1:1000 or 1 µg/ml), 1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid (SKF-89976a; 50 mM; Tocris, Baldwin, Missouri), and 1-(2-tris(4-methoxyphenyl)methoxy)ethyl)-3-piperidinecarboxylic acid ((S)-SNAP 5114; 50 mM; Tocris).

Electrophysiology

On 1–2 days in vitro (DIV), embryonic slice cocultures were transferred to a recording chamber, stabilized by an overlaying platinum ring strung with plastic string mesh, and maintained at 37 °C on a heated stage fit onto an upright microscope (BX50WI, Olympus, Melville, New York). The slice cocultures were perfused at a rate of 0.5 ml/min with oxygenated artificial cerebral spinal fluid (aCSF) containing (in mM) NaCl 124, KCl 5.0, MgCl2 2.0, CaCl2 2.0, glycine 0.01, NaH2PO4 1.25, NaHCO3 26, and glucose 10. In the set of experiments assessing the presence of ambient GABA, the slices were not perfused to avoid possible washout of endogenous GABA, and a constant gentle stream of 95% oxygen/5% CO2 was applied over the surface of the static aCSF. One set of experiments employed acute telencephalic slices derived from E14.5 mouse embryos.

GFP-MGE cells in 1–2 DIV slice cocultures were identified under fluorescence illumination and then visualized under Hoffman Modulation Optics (Modulation Optics, Greenvale, New York) using a 40x water immersion objective (3-mm working distance; Olympus). Real-time images were captured using an analog video camera attached to a video frame grabber board (Integral Technologies, Indianapolis, Indiana) and displayed on a computer monitor, which aided the navigation and placement of the drug and recording pipettes. Patch pipettes were pulled from glass capillaries (1.5-mm outer diameter, 0.86-mm inner diameter; Sutter Instrument Co., Novato, California) to a resistance of 8–10 M{Omega}. Recording pipettes were filled with an internal solution composed of (in mM) KCl 140, CaCl2 1.8, MgCl2 1.0, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 5.0 and supplemented with Mg2+ adenosine triphosphate. Recordings were made using an AxoPatch 200A amplifier (Axon Instruments, Foster City, California). Membrane currents were filtered at 5 kHz, digitized using Clampex v9.0, and analyzed with Clampfit v9.0 (Axon Instruments). Analog signals were monitored online using a chart recorder (Gould, Valley View, Ohio). Statistical analysis was performed using Sigma Stat 3.0 (SPSS Inc., Chicago, Illinois). Mean peak current amplitude of drug-evoked currents was analyzed using Student's t-test. Data were reported as mean ± standard error of the mean (SEM).

One series of experiments used human embryonic kidney (HEK293) cells stably transfected with cDNAs encoding rat {alpha}6/ß2/{gamma}2s GABAA receptor subunits as a bioassay. The HEK293 cell line used in this study ({alpha}6110-34) was a gift from Dr Toshio Narahashi (originally from Dr Donald Carter, Upjohn, Kalamazoo, Michigan). The cells were plated on glass coverslips that were broken into small pieces and maintained in DMEM containing 10% heat-inactivated fetal bovine serum, 2 mM glutamine, penicillin/streptomycin/fungizone, and geneticin (G418). Immediately prior to the experiment, a broken piece of coverslip containing the HEK293 cells was transferred into the recording chamber and onto the surface of the slice coculture near the GE or neocortex. An individual HEK293 cell on the coverslip was identified for whole-cell recording, and the amplitude of current responses to a series of incremental concentrations of GABA (0.01–100 µM) was determined. The same HEK293 cell was then lifted from the coverslip, transferred to the slice coculture, and gently embedded into the tissue. After establishing that GABA responses are preserved, baseline whole-cell current was recorded before, during, and after a 10-s application of 20 µM bicuculline. The amplitude of the bicuculline-induced current was used to extrapolate the concentration of ambient GABA based on the concentration–response curve obtained from that HEK293 cell.

GABA (0.1–200 µM), diazepam (0.5 µM), and bicuculline methiodide (20–50 µM) were dissolved in external solution, stored as frozen stock, and diluted to working concentrations with aCSF immediately prior to each recording session. The drug solutions were loaded into separate barrels of an 8-barrel drug pipette assembly and applied using regulated pulses of pressure (≤ 3 pounds per square inch; Picospritzer, General Valve Corporation, Fairfield, New Jersey) within 10 µm of the soma of the cell under study. Timing and duration of the pressure pulses were controlled by a digital timing unit (Pulse train 1831, WPI). One of the barrels of the multibarrel assembly was routinely filled with aCSF, which was applied between drug applications to clear drugs from the vicinity of the cell. This also served as vehicle control and to control for mechanical artifacts due to bulk flow.

Time-Lapse Video Microscopy

Embryonic slice cocultures (1–2 DIV) were placed in a custom-made mini-incubator designed to maintain a humidified atmosphere saturated with 95% oxygen/5% CO2 and kept at a constant temperature of 37 °C. The mini-incubator was placed on the stage of an upright microscope (BX50WI, Olympus) or a laser scanning confocal microscope (FV300, Olympus). GFP-MGE and CMTMR-labeled cells were visualized using a 10x or a 20x extra long–working distance water immersion lens using the appropriate fluorescence filter. For laser scanning confocal microscopy, slice cultures were scanned using the argon and krypton laser lines of the confocal microscope, and a set of images at a series of depths through the thickness of the slice (z-series) was collected at 5-min intervals to compensate for cells that migrate out of a given plane of focus. Each set of z-series images was then superimposed and flattened to produce a single image that was used to calculate and analyze the rate of migration of GFP-MGE or CMTMR-labeled cells before the CSJ and in the neocortex. The rate of migration of GFP-MGE cells located either before or after the juncture was compared between the control, bicuculline, and diazepam treatment groups using Student's t-test. Data were presented as the mean rate ± SEM.

Immunohistochemistry

Slice cocultures processed with antibody against GFP or activated caspase 3 (casp-3) were fixed overnight using 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), washed with PBS, and blocked for 2 h in 10% normal goat serum (NGS)/0.01% Triton X-100 in PBS. The primary antibody (mouse anti-GFP, 1:1000; Molecular Probes, or rabbit antiactivated casp-3, 1:400; R&D system, Minneapolis, Minnesota) was diluted in 1% NGS/0.01% Triton X-100 in PBS and incubated overnight at 4 °C. Biotinylated goat anti-mouse immunoglobulin G (IgG) (1:200 in PBS; Vector, Burlingame, California) or goat anti-rabbit IgG Alexa Fluor 568 (1:400 in PBS; Molecular Probes) was used as secondary antibody, and slices were incubated at room temperature for 2 h. Slices processed with an antibody against GFP were then incubated in avidin–biotin complex (Elite ABC Kit, Vector), and the reaction product was visualized following preincubation with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Sigma) in 0.05 M Tris hydrochloride–buffered saline followed by incubation in the same medium plus 0.01% hydrogen peroxide.

In double-label experiments employing antibodies against GABA and GFP, slice cocultures were fixed overnight using 0.1% glutaraldehyde/4% paraformaldehyde in PBS. Following washes with PBS, the slices were then resectioned to obtain 20-µm cryosections. The cryosections were blocked for 2 h in 10% NGS/0.01% Triton X-100 in PBS and incubated overnight at 4 °C in primary antibodies adjusted to the appropriate dilution using 1% NGS/0.01% Triton X-100 in PBS (mouse anti-GFP, 1:1000, Novus (Littleton, Colorado); rabbit anti-GABA, 1:1000, Sigma). The cryosections were then incubated for 2 h at room temperature with a cocktail of secondary antibodies (goat anti-rabbit IgG Alexa Fluor 568 (1:400 diluted in PBS; Molecular Probes) and goat anti-mouse IgG Alexa Fluor 488 (1:400 diluted in PBS; Molecular Probes), washed in PBS, mounted onto slides, and coverslipped in FluorSave Reagent (Calbiochem, La Jolla, California).

Crossing Index

Because there is inherent variability from slice to slice with regard to the size of the GFP-MGE explant and, thus, the number of migrating cells, we devised a "crossing index" (i.e., crossing the CSJ) to normalize and quantify the data. Slice cocultures processed for GFP immunostaining were examined on an upright microscope (BX60; Olympus), and images were taken using a Spot camera (2.2.0; Diagnostic Instruments, Sterling Heights, Michigan). Montages of individual slice cocultures were constructed to include a full view of the MGE and neocortex. The cortex distal to the CSJ (referred to as AJ) was divided into five 100-µm-wide bins (see Fig. 6A). A 200-µm-thick area immediately adjacent and proximal to the CSJ was denoted as "before juncture" (see Fig. 6A). Using Image J (National Institutes of Health, Bethesda, Maryland), immunopositive cells were counted in each bin, and crossing index was calculated as the number of cells in a given bin defined distal to the CSJ divided by the number of cells before the CSJ. Crossing index was compared between different treatment groups within given bins using one-way analysis of variance and Holm–Sidak method. Data were presented as mean ± SEM.


Figure 6
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  		Figure 6. Modulators of GABAA receptor function regulate migration of MGE-derived cells (II). (A) A slice coculture containing immunostained GFP-MGE cells illustrating the bins proximal (BJ) and distal (AJ) to the CSJ used to calculate crossing index. (B) Crossing index of slice cocultures treated without and with Bicu (20 µM), diazepam (0.5 µM), GABA (1 µM), GABA (1 µM) + Bicu (20 µM), an antibody against GABA (1:1000), SKF-89976a (50 µM), and SNAP 5114 (50 µM). Data were expressed as mean ± SEM. For each set of data calculated at a given distance from the CSJ, the asterisks denote statistically significant differences from control (Student's t-test; P < 0.05). AJ, after juncture; Bicu, bicuculline; BJ, before juncture.

 
Reverse Transcription–Polymerase Chain Reaction

Time-pregnant mice were sacrificed at E13.5–E14.5 by CO2 asphyxiation, and the embryos were removed by cesarean section. Whole embryonic brains were isolated and a paramidsagittal incision was made bilaterally along the dorsal rostral-to-caudal extent of the cortex, thereby splaying the cortical mantle and revealing the V-shaped ridges of the LGE and MGE on the floor of the lateral ventricles. The MGE and neocortex were dissected and processed separately to obtain profiles of candidate GABAA receptor subunit mRNAs employing an RT-PCR–based protocol modified from that published previously (Yeh and others 2002Go). Briefly, tissue derived from MGE and neocortex was homogenized in TRI reagent (Molecular Research Center Inc., Cincinnati, Ohio), and RNA was extracted using bromochloropropane, precipitated by adding isopropanol, ammonium acetate (3 N), and glycogen (5 mg/ml), washed in 75% ethanol, and then solubilized in ribonuclease-free water. First-strand cDNA was synthesized by the addition of reverse transcriptase (RT-SSIII, Invitrogen), 5x first-strand buffer, 2.5 mM deoxynucleoside triphosphates (dNTPs), 300 ng Oligo (dT) (Integrated DNA Technologies, Coralville, Iowa), RNasin inhibitor, and 100 mM dithiothreotol in a final volume of 30 µl and incubated for 1 h at 42 °C. Polymerase chain reaction amplification of the reversed transcribed cDNA template was then performed using a programmable thermocycler (I-cycler, BioRad) in a solution containing thermal buffer (10x Taq polymerase buffer without MgCl2; Invitrogen), 2.5 mM MgCl2, 0.25 mM dNTPs, 2 U Taq DNA polymerase (Invitrogen), 50 pmol of GABAA receptor subunit–specific primers, and 1 µl cDNA. "No-RT" controls were routinely run with water added in lieu of the sample. A 15-µl aliquot of the reaction product was then electrophoresed parallel to a molecular weight ladder through 1.5% agarose and visualized under UV illumination after staining with ethidium bromide. Table 1 lists the GABAA receptor subunit–specific primer sequences used (Liu and Burt 1998Go). ß-Actin was routinely included as a positive control and water as a negative control.


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  		Table 1 GABAA subunit and ß-actin primer sequencesa

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The present study employed an isochronic and isotopic slice coculture system, in which GFP-MGE explants from E13.5–E15.5 GFP-expressing mice were incorporated into the MGE of coronal telencephalic slices derived from wild-type littermates (Fig. 1A1,A2). Beyond 1 day in culture, GFP-MGE cells migrated along a tangential trajectory from the MGE to the neocortex (Fig. 1B). The GFP-MGE cells integrated well into the wild-type host slices because they comigrated at a similar rate (P = 0.52) with the endogenous cells labeled with the cell tracker CMTMR (Alifragis and others 2002Go) placed in the MGE (Fig. 1C–E).

An Ambient Level of GABA Is Present along the Tangential Migratory Path

A prerequisite to testing the hypothesis that GABA regulates tangential migration is demonstrating its ambient presence at a level that is sufficient to exert a tonic influence on cells in the neocortex and MGE. GFP-MGE cells along the tangential migratory path in 1–2 DIV slice cocultures were identified for whole-cell recording (Fig. 2A1,A2). Under voltage clamp conditions, the baseline current was monitored before, during, and after a focal 10-s exposure to bicuculline (20 µM). In IZ of the developing neocortex, exposing GFP-MGE cells to bicuculline consistently uncovered an outward shift in the baseline current (12 ± 1.1 pA; 15 of 19 cases; Fig. 2B1). By contrast, bicuculline did not cause a shift in baseline current in the majority of cells monitored in the MGE (12 of 15 cases; Fig. 2B2). In the remaining 3 cells recorded in the MGE, exposure to bicuculline shifted baseline current 6.7 ± 1.3 pA, which was significantly lower than that observed in cells monitored in the IZ (P < 0.01; Fig. 2E).


Figure 2
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  		Figure 2. The concentration of ambient GABA in the developing neocortex and MGE is approximately 0.5 µM. (A) GFP-MGE cell recorded in the IZ of the neocortex visualized under epifluorescence (A1) and Hoffman Modulation Contrast Optics (A2), respectively. In (A2), the patch clamp pipette used to monitor whole-cell current and the multibarrel drug pipette assembly used to apply drugs converge on the cell being recorded from the right and left, respectively. Scale bar in A1 = 10 µm. (B1) Application of 20 µM bicuculline (Bicu) to a cell in the IZ uncovered reversibly a 15-pA outward current and a concomitant decrease in baseline noise. (B2) Application of the same concentration of Bicu to a cell in the MGE did not produce an outward current. (C1) HEK293 cell being recorded and tested for sensitivity to GABA delivered via a multibarrel drug pipette assembly shown at the left. The cell was subsequently lifted from and embedded in the MGE to monitor the amplitude of the Bicu-induced outward current (C2). Scale bar in (C1) = 10 µm. (D) Concentration–response curve for HEK293 cells stably expressing {alpha}6/ß2/{gamma}2s recombinant GABAA receptors. The inset illustrates a series of current responses of an HEK293 cell to varying and incremental concentrations of GABA that are used to construct the concentration–response curve. (E) Mean amplitude of the Bicu-induced outward current from cells recorded in the neocortex (Cortex), MGE, and HEK293 cells placed in the MGE (HEK-MGE). Asterisks denote a statistically significant difference compared with control (Student's t-test; P < 0.01). (F) Estimates of ambient GABA concentration (mean ± SEM) derived from data obtained from cells recorded in the IZ of 2 DIV slice cocultures (Cortex (2 DIV)), HEK293 cells placed in the MGE of 2 DIV slice cocultures (HEK-MGE), and IZ cells from acutely prepared telencephalic slices (Cortex (acute)). The results reveal comparable concentrations of ambient GABA (analysis of variance; P = 0.25).

 
Although the incidence of detecting a bicuculline-induced outward current in the MGE was low, it nonetheless implied the presence of an ambient level of GABA. Given that the bicuculline-induced current shift was more prominent in the neocortex, we postulated that either the ambient level of GABA was higher there or cells in the MGE were less responsive to the same ambient level of GABA, or both. To verify the presence and optimize electrophysiological detection of ambient GABA in the MGE, HEK293 cells stably expressing functional {alpha}6/ß2/{gamma}2s recombinant GABAA receptors and grown on broken pieces of glass coverslips were added to the slice cocultures (see Materials and Methods). Concentration–response curves were first established for individual HEK293 cells grown on coverslips (Fig. 2C1) by monitoring the amplitude of whole-cell current responses to a series of incremental GABA concentrations (0.01–100 µM; mean 50% effective concentration [EC50] = 2.5 µM, Fig. 2D; n = 13). Each HEK293 cell was then gently lifted and placed in the MGE (Fig. 2C2) to monitor changes in whole-cell current as a result of bicuculline application. As summarized in Figure 2E, application of bicuculline in the vicinity of the HEK293 cells produced an outward current (31.6 ± 4.8 pA; n = 16) that, consistent with the high-affinity binding profile reported for {alpha}6 subunit–containing recombinant GABAA receptors (Zheng and others 1994Go; Knoflach and others 1996Go; Saxena and Macdonald 1996Go), was significantly larger than that observed in cells native to the MGE (P < 0.01). The results of this experiment effectively confirmed the tonic presence of GABA in the MGE.

We determined the concentration of ambient GABA in the extracellular milieu of the neocortex and MGE. In each cell, the amplitude of the bicuculline-induced current was fit to the GABA concentration–response profile determined for that cell to derive at an estimate of the ambient GABA concentration. Such analyses revealed that the developing neocortex contained 0.5 ± 0.1 µM of ambient GABA (Fig. 2F). Based on data derived from recombinant GABAA receptor–expressing HEK293 cells, the ambient concentration of GABA in the MGE was 0.4 ± 0.1 µM, which was not significantly different from that estimated to be in the neocortex (P = 0.25; Fig. 2F). Finally, to rule out the possibility that this ambient level of GABA was an artifact of having maintained the slice cocultures long term in vitro, we established that the same concentration of ambient GABA (0.5 ± 0.1 µM) was present in the neocortex of acutely prepared 250-µm telencephalic slices derived from E14.5 mouse embryos (Fig. 2F).

A potential source of ambient GABA may be the MGE-derived cells themselves because they are destined to comprise a prominent population of GABAergic cortical interneurons (DeCarlos and others 1996Go; Anderson and others 1997Go, 1999Go, 2001Go; Tamamaki and others 1997Go; Lavdas and others 1999Go; Wichterle and others 2001Go; Nery and others 2002Go; Rakic and Zecevic 2003Go; López-Bendito and others 2004Go; Xu and others 2004Go). Antibodies against GABA and GFP revealed GABA-immunopositive GFP-MGE cells as well as endogenous cells in the MGE, neocortex, and along the tangential migratory route in between (Fig. 3). Consistent with earlier reports (Van Eden and others 1989Go; Tamamaki and others 1997Go; Lavdas and others 1999Go; Anderson and others 2001Go; Jimenez and others 2002Go; Polleux and others 2002Go), GABA-immunoreactive cells were abundant in the embryonic neocortex (Fig. 3A), being concentrated along the SVZ, IZ, and MZ. Our analysis revealed that, in 2 DIV telencephalic slice cocultures derived from E14.5 brain, the percentage of GABA-immunoreactive GFP-MGE cells was significantly higher in the neocortex than in the MGE (39 ± 2.3% vs. 9 ± 0.8%, respectively; P < 0.001; data not shown). Nonetheless, these results suggest that GABA synthesized by and presumably released from migrating MGE-derived cells could contribute to the maintenance of an ambient extracellular level of GABA in the MGE and neocortex. Thus, the failure to demonstrate consistently a bicuculline-induced outward current in the MGE is not likely due to an absence of ambient GABA but more likely due to other factors, such as regionally dependent responsiveness of migrating MGE-derived cells to GABA.


Figure 3
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  		Figure 3. GABA-immunoreactive GFP-MGE cells increase with migration into the neocortex. (A) GABA-immunoreactive cellular profiles in the neocortex (upper panel) and MGE (lower panel) of E14.5 telencephalic sections. Note that immunostaining for GABA is clearly evident in the cells in the MZ, IZ, and SVZ of the neocortex but weak and diffuse in the MGE. Arrowheads point to examples of GABA-immunoreactive cells. Scale bar = 100 µm. (B) Cells immunopositive for GFP (green) and GABA (red) in the MGE, CSJ, and IZ of the neocortex seen at higher magnification in 20-µm sections resectioned from 2 DIV E14.5 telencephalic slice cocultures. Arrows in the right bottom panel points to examples of cells that are colabeled with GFP and GABA. Scale bar = 10 µm.

 
MGE-Derived Cells Express Functional GABAA Receptors

GFP-MGE cells migrating away from the MGE, in the region of the CSJ and in the IZ of the neocortex, were identified for whole-cell recording to test their responsiveness to GABA. Those recorded in the MGE and the CSJ region (Fig. 4A) typically displayed low-amplitude currents (18.9 ± 3.6 pA) in response to focally applied GABA (100–200 µM) that were completely blocked by bicuculline (Fig. 4B; n = 21). In IZ of the same slice cocultures (Fig. 4C), the same concentration of GABA evoked responses in all the GFP-MGE cells examined (35 cases) with a mean amplitude of 94.0 ± 16.2 pA (Fig. 4D). Although significantly higher than that recorded from cells in the MGE region, the mean amplitude of the GABA response of cells monitored in the IZ was nonetheless modest as lower concentrations of GABA routinely evoked robust (>200 pA) bicuculline-sensitive responses in nonpyramidal cortical neurons recorded in slices derived from postnatal mice (Fig. 4E,F).


Figure 4
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  		Figure 4. Differential expression of functional GABAA receptors and GABAA receptor subunit mRNAs in E14.5 MGE and neocortex. (A) A migrating GFP-MGE cell in the GE/CSJ area of a 1 DIV E14.5 telencephalic slice coculture. (B) The GABA (100 µM)-activated response of the cell in (A) was blocked reversibly by Bicu (50 µM). (C) Fluorescence image of a migrating GFP-MGE cell within the same slice coculture that has reached the IZ of the neocortex. Arrow indicates the pial surface. (D) Response of the cell in (C) to GABA (100 µM). A nonpyramidal cell in Layer 1 of a postnatal day 5 cortical slice (E) displays a robust GABA response that was blocked reversibly by 50 µM Bicu (F). RT-PCR profiling of GABAA receptor subunit mRNAs in E14.5 MGE (G) and neocortex (H). ß-Actin (ß-act) was used as a positive control. No-RT controls were routinely run in parallel (not shown). The reaction product was electrophoresed parallel to a MW ladder. Arrowhead indicates the 600-bp mark on the ladder. Bicu, bicuculline; CTX, cortex; MW, molecular weight.

 
The whole-cell membrane capacitance (Cm) measured in migrating GFP-MGE cells recorded proximal to the CSJ and in the neocortex remained constant (4.2 ± 1.9 pF, n = 7; 4.3 ± 1.9 pF, n = 11, respectively; P = 0.68). Thus, the finding that GFP-MGE cells progressively acquire responsiveness to GABA along the tangential migratory path reflects an increase in current density (pA/Cm) and suggests that MGE-derived cells either acquire more GABAA receptors or express subunits that assemble into GABAA receptor isoforms that are more sensitive to GABA, or both. With specific regard to the possibility of differential subunit expression, RT-PCR–based profiling of the {alpha}, ß, and {gamma} family of GABAA receptor subunit mRNAs ({alpha}1–6, ß1–3, and {gamma}1–3) revealed partially overlapping expression patterns in tissue samples microdissected from the MGE and neocortex at E14.5 (Fig. 4G,H). Although amplicons corresponding to a number of GABAA receptor subunit transcripts were evident in the MGE ({alpha}3–5, ß1–3, and {gamma}1) and neocortex ({alpha}1–5, ß1, ß3, and {gamma}1–3), certain transcripts appeared to be unique to the MGE (ß2) or neocortex ({alpha}1, {alpha}2, {gamma}2, and {gamma}3). Thus, assembly of different cadres of subunits into GABAA receptor isoforms that vary in functional properties may contribute to the differential responsiveness to GABA observed in the tangentially migrating MGE-derived cells.

GABAA Receptor Modulators Affect the Migration of MGE-Derived Cells

If ambient GABA were to regulate tangential migration via GABAA receptor activation, then manipulating GABAA receptor function should affect tangential migration. GFP-MGE explants incorporated into the MGE of E14.5 embryonic slices were maintained in culture for 2 DIV in the presence or absence of either bicuculline (20 µM) or diazepam (0.5 µM), pharmacological agents that block or potentiate GABAA receptor function, respectively. By 2 DIV, compared with the control condition (Fig. 5A,D), exposure to bicuculline led to impeded cortical entry, with GFP-MGE–derived neurons accumulating at the CSJ; very few cells passed this juncture to enter the cortex (Fig. 5B,E). The impeding effect of bicuculline was not due to cell death, based on 2 observations. First, parallel immunostaining of bicuculline-treated and control slice cocultures with an antibody against activated casp-3 revealed no significant difference in the density of immunopositive cells along the migratory path (Student's t-test; MGE: P = 0.65, CSJ: P = 0.42, cortex: P = 0.40). In addition, migration resumed upon replenishing the slice cocultures with either control medium or medium containing diazepam (data not shown). By contrast, more GFP-MGE cells transgressed the CSJ in cocultures exposed to diazepam (Fig. 5C,F), and they migrated to further extents in the cortex. Thus, blocking GABAA receptor function impeded migration of MGE neurons into the neocortex, whereas enhancing GABAA receptor function had the reciprocal effect. Within the neocortex, differences in the migration pattern of GFP-MGE cells were also evident, although these have yet to be analyzed quantitatively. Overall, these observations suggest that ambient GABA regulates the tangential migration of MGE-derived neurons.


Figure 5
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  		Figure 5. Modulators of GABAA receptor function regulate migration of MGE-derived cells (I). (A) At 2 DIV, GFP-MGE cells migrate past the CSJ to enter the neocortex. (B) Treatment of sister cocultures with bicuculline (20 µM) resulted in fewer GFP-MGE cells crossing the CSJ. (C) Treatment with diazepam (0.5 µM) enhanced migration of GFP-MGE cells throughout the extent of the neocortex. (D), (E), and (F) are enlargements of the respective boxed areas in (A), (B), and (C). Arrows indicate GFP-MGE explant placement. Images (A–C) were taken at the same magnification. Scale bar in (C) = 200 µm. Images (D–F) were taken at the same magnification. Scale bar in (F) = 100 µm.

 
A crossing index (i.e., crossing the CSJ) was devised to normalize the variability with regard to the size of the GFP-MGE explants and, thus, the number of migrating cells (Fig. 6A; see also Materials and Methods). Figure 6B indicates that, within the first 300 µm in the AJ region, crossing index was significantly lower in the bicuculline-treated, and higher in the diazepam-treated, than that in control slice cocultures. Inclusion of GABA (1 µM) in the culture medium mimicked the diazepam-induced effect, which was blocked by concomitant exposure to bicuculline (Fig. 6B). On the other hand, treatment with an antibody against GABA (1 µg/ml) mimicked the bicuculline-induced change in the crossing index (Fig. 6B). The effectiveness of treatment with this concentration of antibody was demonstrated by results of a control experiment in which the bicuculline-induced shift in baseline whole-cell current that was typically evident when recording GFP-MGE cells located in the neocortex (e.g., Fig. 2B) was absent in neocortical cells (10 of 10 cases) recorded in slice cocultures that had been treated for 24–36 h with medium containing 1 µg/ml GABA antibody (data not shown). GABA (100 µM) elicited current responses in all these cells, confirming the expression of GABAA receptors. Thus, conditions that alter the level of ambient GABA or modulate the function of GABAA receptors affect the number of GFP-MGE cells that migrate into the cortex.

We next asked whether activity of GABA transporters (GAT) may contribute to modulating the level of ambient GABA. In E14 mouse, GAT-1 mRNA is present in the LGE and MGE, whereas both GAT-1 and mGAT-4 (or rGAT-3) mRNAs are present in the cortex (Evans and others 1996Go; reviewed in Conti and others 2004Go). E14.5 slice cocultures were maintained for 48 h in medium supplemented with SKF-89976a (50 µM), a GAT-1–selective inhibitor, or (S)-SNAP 5114 (50 µM), a GAT inhibitor selective for GAT-2 and GAT-3. Counts of migrating GFP-MGE cells in control and slice cocultures treated with either GAT inhibitors revealed no significant difference in the crossing index (Fig. 6B). These results suggest that GABA reuptake mechanisms do not play a prominent role in regulating tangential cell migration.

We determined whether bicuculline or diazepam exposure affected the rate of migration of the GFP-MGE–derived cells by time-lapse video microscopy (Fig. 1D). The rate of migration (45 ± 12.5 µm/h), derived by measuring the distance traversed by a given cell between successive images as a function of time for up to 3 h, with images taken once every 5 min, was analyzed in 7 control slice cocultures (54 cells total), 6 from the bicuculline-treated group (51 cells total), and 3 from the diazepam-treated group (14 cells total), representing 3 separate experiments. Only individual GFP-MGE cells that migrated forward and along a plane tangential to the slice were analyzed; data derived from those with leading processes oriented in the reverse direction or that traversed vertically through the thickness of the slice were excluded from this study. No significant difference in migration rate was found in individual cells between the experimental groups either before or after the CSJ (P = 0.74; Fig. 7A), in agreement with a previous report (Polleux and others 2002Go). Nonetheless, when analyzed as a population, Figure 7B illustrates that there was a significant difference (P < 0.002) in the ratio of GFP-MGE cell counts in 400 x 100–µm areas around the CSJ area to those in the MGE between the control (0.63 ± 0.12) and the bicuculline-treated (1.56 ± 0.18) groups. These results confirm the accumulation of GFP-MGE cells around the CSJ of slice cocultures exposed to bicuculline and suggest that rate of migration, per se, cannot fully account for the differences observed in tangential migration into the neocortex under the different experimental conditions.


Figure 7
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  		Figure 7. Treatment with GABAA receptor modulators does not affect the migration rate of GFP-MGE cells but results in accumulation of cells proximal to the CSJ. (A) Migration rate of individual cells was measured using real-time confocal video microscopy before and after the CSJ in 2 DIV slice cocultures without (Ctl) and with 20 µM bicuculline (Bicu) and 0.5 µM diazepam (DZ) treatment. Data were expressed as mean ± SEM. (B) Comparison of "accumulation index," reflecting the ratio of the number of GFP-MGE cells in defined areas (400 x 100 µm) around the CSJ to that in the MGE, revealed a significant buildup of GFP-MGE cells in the vicinity of the CSJ under conditions of exposure to Bicuculline versus control (P < 0.002).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study tested the hypothesis that ambient GABA, via tonic activation of GABAA receptors, regulates the migration of MGE-derived cells into the developing neocortex. The GFP-MGE slice coculture system was well suited for studying tangential migration; the GFP-MGE cells were readily visualized and followed in real time, migrated at the same rate and along similar paths as their endogenous counterparts, and were amenable to patch clamp recording and immunohistochemistry. The major findings of this study are 1) an ambient level of GABA is present in the MGE and neocortex of the embryonic brain at a concentration of 0.5 µM, 2) MGE-derived cells express functional GABAA receptors that are tonically activated along the tangential migratory path by ambient GABA, 3) the receptivity of MGE-derived cells to GABA increases with migration, and 4) pharmacological manipulations that block or enhance GABAA receptor function impede or promote, respectively, the migration of MGE-derived cells into the cortex.

Ambient GABA Tonically Activates GABAA Receptors in the MGE and Developing Neocortex

Tonic activation of GABAA receptors by GABA has been proposed in the rodent hippocampus and cortical plate during development (Owens and others 1999Go; Demarque and others 2002Go; Manent and others 2005Go; reviewed in Farrant and Nusser 2005Go). In this study, an ambient GABA-mediated influence was demonstrated in the MGE and neocortex much earlier in embryonic development, specifically, during the height of tangential migration (E14.5–E15.5). This places GABA chronologically and in a favorable position to play a regulatory role in corticogenesis.

In the neocortex, focal application of bicuculline revealed an outward current accompanied by a decrease in baseline noise. In studies that have used this pharmacological approach to show tonic activation of GABAA receptors in various regions of the developing brain (Owens and others 1999Go; Demarque and others 2002Go; Rossi and others 2003Go; Caraiscos and others 2004Go; Manent and others 2005Go), paracrine release of GABA has been postulated, and its ambient accumulation has been attributed to GABA reuptake mechanisms being either undeveloped or functionally immature in the embryonic brain (Evans and others 1996Go; Jursky and Nelson 1996Go, 1999Go; reviewed in Conti and others 2004Go). Our results indicate that disrupting GAT function using SKF-89976a or (S)-SNAP 5114 did not alter the pattern of tangential migration in the telencephalic slice cocultures, pointing to an interplay between ambient GABA and GABAA receptors in regulating the migration of MGE-derived cells that is independent of GABA reuptake mechanisms.

To demonstrate the presence of ambient GABA in the MGE, we devised a bioassay to optimize the electrophysiological detection of tonic GABAA receptor activation, using HEK293 cells stably expressing recombinant {alpha}6/ß2/{gamma}2s GABAA receptors as surrogate to native MGE cells. Recombinant {alpha}6/ß2/{gamma}2s GABAA receptors display exquisite sensitivity to GABA, with EC50 ranging from 0.1 to 3 µM (Zheng and others 1994Go; Knoflach and others 1996Go; Saxena and Macdonald 1996Go). When HEK293 cells expressing {alpha}6/ß2/{gamma}2s recombinant GABAA receptors were placed in the MGE, application of bicuculline consistently produced an outward current. Thus, our bioassay uncovered an ambient level of GABA in the MGE and yielded results that could not have been easily resolved using conventional experimental approaches.

In both MGE and neocortex, the concentration of ambient GABA is approximately 0.5 µM. Such a low concentration of GABA tonically activates but does not desensitize GABAA receptors, conceivably maintaining a baseline level of receptor activity in MGE-derived cells that can be up- or downregulated by fluctuating levels of ambient GABA along the tangential migratory path. This notion is in line with investigations of GABAA receptor kinetics in which low agonist concentrations (0.5–2 µM) were employed to achieve prolonged monitoring of steady-state receptor activation without desensitization (Macdonald and others 1989Go; Twyman and others 1989Go; Twyman and Macdonald 1992Go; Rogers and others 1994Go).

GFP-MGE Cells Increase Receptivity to GABA with Tangential Migration

Certain GABAA receptor isoforms are insensitive to blockade by bicuculline (Polenzani and others 1991Go; Shimada and others 1992Go; Woodward and others 1993Go; Yeh and others 1996Go; reviewed in Johnston 1986Go), and some of these are transiently expressed during development (Strata and Cherubini 1994Go; Martina and others 1995Go). However, the expression of such receptors cannot account for the failure to demonstrate a bicuculline-induced outward current in the majority of cells sampled in the MGE because GABA responses were blocked by the concurrent application of bicuculline. A more likely explanation may be the disposition of GABAA receptors, either in terms of density or differential makeup of the receptor subunits, or both. This is corroborated by 2 findings reported in this study: 1) GFP-MGE cells in the vicinity of the MGE showed lower receptivity to GABA relative to those in the IZ of the neocortex and 2) RT-PCR–based profiling of GABAA receptor subunit transcripts revealed overlapping yet different expression patterns in the MGE vis-à-vis the neocortex.

Our results suggest that tangentially migrating GFP-MGE cells become more sensitive to GABA as they migrate from the GE to the neocortex. During development of the central nervous system, GABAA receptor subunit expression changes during migration, differentiation, and synaptogenesis (Laurie and others 1992Go; Poulter and others 1992Go, 1993Go; Ma and others 1993Go). GABA itself has been shown to regulate the expression of GABAA receptors subunits (Poulter and others 1997Go). The present study demonstrated the expression of different complements of GABAA receptor subunit transcripts in the MGE vis-à-vis the neocortex. For example, the {alpha}1 subunit transcript revealed in the neocortex was absent in the MGE. Because the {alpha} subunits regulate GABAA receptor–mediated desensitization and deactivation (Gingrich and others 1995Go; Tia and others 1996Go; Lavoie and others 1997Go; McClellan and Twyman 1999Go; Bianchi and others 2002Go), and are developmentally regulated (Laurie and others 1992Go), their differential expression suggests that GABAA receptors expressed in MGE-derived cells mature in functional properties as the cells migrate tangentially into the neocortex. Furthermore, the finding that exposure to diazepam modulated the entry of MGE-derived cells into the neocortex (crossing index) points to MGE-derived cells expressing GABAA receptor {gamma} subunits (Pritchett, Luddens, and others 1989Go; Pritchett, Sontheimer, and others 1989Go). Indeed, diazepam (0.5 µM) potentiated the amplitude of GABA-activated current responses in MGE-derived cells (data not shown). These results formulate the basis for future investigations to delineate the profile of sensitivity to GABA with that of GABAA receptor subunit expression in individual MGE-derived cells.

GABAA Receptor Modulators Affect Cortical Entry of Tangentially Migrating MGE-derived Cells

Studies examining the effect of GABA receptor modulators on the migration of immature cortical neurons and neuronal precursor cells have yielded inconsistent results. For example, the GABAB receptor agonist baclofen and antagonist saclofen were reported to facilitate and retard cortical neuronal migration, respectively (Behar and others 1996Go; López-Bendito and others 2003Go). Although these studies did not demonstrate the presence of the subunits required to form a functional dimeric GABAB receptor (Robbins and others 2001Go), the results were consistent with a facilitating effect of endogenous GABA. On the other hand, ambient GABA has been reported to suppress the migration of neuronal precursor cells from postnatal and adult SVZ, an effect that could be released upon exposure to bicuculline (Bolteus and Bordey 2004Go). In addition, there is evidence that GABA inhibits migration of cortical cells either via an unidentified G protein–coupled picrotoxin-sensitive receptor or a more conventional bicuculline-sensitive GABAA receptor (Behar and others 1998Go, 2000Go). In the least, these apparently discrepant results could be accounted for by the type and concentration of GABA receptor modulators used as well as differences in the population and developmental ages of migrating neurons examined. Nonetheless, a basic assumption made in all of these studies is the presence of an undetermined ambient level of GABA. Based on the results presented here, our contention is 2-fold: first, that a GABA-mediated effect on tangential migration should be investigated at a physiologically relevant concentration of ambient GABA (0.5 µM), and second, that expression of GABAA receptors dictates the manifestation of this effect.

A basic premise of our study was that demonstrating experimentally the presence of ambient GABA and the expression of functional GABAA receptors in MGE-derived cells were prerequisites to testing the hypothesis that tonic activation of GABAA receptors regulates tangential migration. This was revealed by analyzing the pattern and extent of tangential migration of GFP-MGE cells in telencephalic cocultures exposed to agents known to affect GABA–GABAA receptor interaction. Specifically, addition of an antibody against GABA, which sequesters endogenous GABA, and bicuculline, which blocks GABAA receptor activation, impeded entry of GFP-MGE cells into the developing neocortex. Augmenting GABAA receptor function by exposure to diazepam promoted cortical entry of GFP-MGE cells, as did exogenous addition of GABA, which was prevented in the presence of bicuculline. These experiments employed bicuculline (20–50 µM) and diazepam (0.5 µM) in concentrations that antagonized and potentiated, respectively, GABAA receptor–mediated current responses in our previous studies (Sapp and Yeh 2000Go; Chan and Yeh 2003Go; Cheng and Yeh 2005Go). Overall, although the mechanism remains to be elucidated, the results of this study indicate that GABA provides an ambient milieu that facilitates passage of MGE-derived cells into the developing neocortex.

Two additional observations are noteworthy. First, the migration of MGE-derived cells from their origin to the CSJ was unaffected by any of the experimental manipulations, as best exemplified in this study by treatment of telencephalic slice cocultures with bicuculline. Because MGE-derived cells located proximal to the CSJ displayed modest GABA-activated current responses that increased in amplitude as migration continued into the neocortex, a parsimonious hypothesis to account for the lack of a bicuculline-induced effect prior to the CSJ is that the MGE-derived cells mature in their expression and disposition of functional GABAA receptors. This adds a cell-autonomous maturational component to complement the exogenous short-range or contact-dependent cues reported to direct or restrict tangential migration (Neyt and others 1997Go; Marín and others 2003Go). Second, even in the presence of bicuculline, a small subset of GFP-MGE cells migrated past the CSJ and into the developing neocortex, suggesting that these cells do not express GABAA receptors, express them at low levels, or express GABAA receptor isoforms that are insensitive to blockade by bicuculline. These observations will need to be extended by determining whether MGE-derived cells along the tangential migratory route are heterogeneous in terms of GABAA receptor pharmacology and whether there is differential expression of receptor density and/or GABAA receptor subunit profiles in the course of their maturation.


   Acknowledgments
 
This study was supported by Public Health Service grant RO1 MH069826. The authors thank Dr Roman Giger for critical reading of the manuscript.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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