GABA Receptor Antagonists Modulate Postmitotic Cell Migration in Slice Cultures of Embryonic Rat Cortex

Toby N. Behar, Anne E. Schaffner, Catherine A. Scott, Carolyn L. Greene and Jeffery L. Barker

Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Recent studies indicate that GABA acts as a chemoattractant during rat cortical histogenesis. In vivo, GABA localizes in appropriate locations for a chemoattractant, along migratory routes and near target destinations for migrating cortical neurons. In vitro, GABA induces dissociated embryonic cortical neurons to migrate. Here, embryonic rat cortical slices were cultured in the presence or absence of GABA receptor (GABA-R) antagonists to assess GABA's effects on neuronal migration in situ. Gestational day 18 (E18) cortical slices were incubated overnight in bromodeoxyuridine (BrdU)-containing medium to label ventricular zone (vz) cells as they underwent terminal mitosis. The slices were then cultured in BrdU-free medium with or without GABA-R antagonists. In control slices, most BrdU+ cells were observed in the cortical plate (cp) after 48 h. In contrast, cultures maintained in either saclofen (a GABAB-R antagonist) or picrotoxin (a GABAA/C-R antagonist) had few BrdU-labeled cp cells. However, the effects of the two antagonists were distinct. In the picrotoxin-treated slices, nearly half of all BrdU+ cells remained in the vz and subventricular zone (svz), whereas saclofen treatment resulted in an accumulation of BrdU+ cells in the intermediate zone (iz). Bicuculline, a GABAA-R antagonist, did not block, but rather enhanced migration of BrdU+ cells into the cp. These results provide evidence that picrotoxin-sensitive receptors promote the migration of vz/svz cells into the iz, while saclofen-sensitive receptors signal cells to migrate into the cp. Thus, as cortical cells differentiate, changing receptor expression appears to modulate migratory responses to GABA.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
During cortical development, neuroblasts undergo terminal mitosis in proliferative regions adjacent to the ventricle, then migrate into the cortical plate (Jacobson, 1991Go). Migrating cortical neurons have been observed to move both radially and tangentially (Rakic, 1972Go; Hatten, 1990Go; Rakic, 1990Go; O'Rourke et al., 1992Go, 1995Go). Recent studies suggest that during development, chemoattractants provide directional cues to the migrating cells. Some molecules expressed near migrating neurons in vivo stimulate nerve cell movement in vitro (Komuro and Rakic, 1993Go; Behar et al., 1997Go, 1998Go). Among these, {gamma}-amino butyric acid (GABA) appears to be a likely candidate for a chemoattractant. During the final week of gestation, GABA is transiently expressed near the target destinations of migratory neurons (Lauder et al., 1986Go; Behar et al., 1996Go). The release of GABA from cells in these regions may stimulate neuronal migration in vivo. GABA acts as a chemoattractant for immature neurons in vitro, inducing both directed migration (chemotaxis) and random motility (chemokinesis) of acutely dissociated embryonic cortical cells (Behar et al., 1996Go, 1998Go).

In vitro studies on cortical dissociates revealed that low GABA concentrations (femtomolar) promote chemotaxis of immature neurons dissociated from ventricular/subventricular regions (vz/svz), while micromolar GABA evokes chemokinesis of more mature neurons isolated from the cortical plate/subplate (cp/sp) (Behar et al., 1996Go, 1998Go). Pharmacological studies indicate that the motility-promoting effects of GABA involve multiple classes of receptors. Baclofen (a GABAB-R agonist) and CACA (a GABAC-R and metabotropic GABA-R agonist) (Kerr and Ong, 1992Go; Feigenspan et al., 1993Go; Zhang et al., 1997Go) mimic the chemotropic effects of GABA on dissociated cells in vitro (Behar et al., 1996Go), while the receptor antagonists saclofen and picrotoxin (Kerr and Ong, 1992Go; Jackel et al., 1994Go) block motility, indicating that GABA stimulates migration via multiple classes of GABA receptors.

The studies using cortical dissociates involve exposing cells to exogenously applied GABA. To determine if GABA released from cortical cells in situ influences motility of embryonic neurons, we analyzed the effects of different GABA receptor (GABA-R) antagonists on cellular migration in cultured slices, in the absence of exogenously applied GABA. The cultured slices provide a system in which cellular migration can be observed in a setting that maintains much of the anatomical organization of the tissue and integrity of cell-to-cell interactions. Tissue slices can be cultured under conditions in which they remain organo-typically organized for weeks, and under appropriate conditions, the major cell types are present and their basic biophysical properties remain intact (Caeser et al., 1989Go).

In the present study, proliferating cells in the vz/svz of cultured slices were labeled with bromodeoxyuridine (BrdU), which is incorporated during DNA synthesis. We then followed the migratory patterns of the BrdU+ neurons under control conditions and in the presence of antagonists at different GABA-Rs. Our pharmacological results in the slices replicate those obtained with dissociated cells (Behar et al., 1996Go, 1998Go), providing evidence that endogenous GABA promotes cortical neuronal migration via activation of multiple GABA-Rs.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Migration of embryonic day (E) 18 cortical cells was used in the present study. At this stage, midway through the lamination process, most migrating cells are neurons (Bayer, 1990Go; Jacobson, 1991Go).

In Vivo BrdU-labeling of Proliferating Cells

Pregnant (E18) rats were injected i.p. with a single dose BrdU at 50 µg/g body wt to label proliferating cells in vivo. Four hours or 3 days post-injection the embryos were collected by Cesarean section and the brains were removed. Cortices were sectioned into 300 µm coronal slices on a vibratome and were immediately fixed for 1 h in 4% para-formaldehyde (PF), then stained with anti-BrdU antiserum as described below. Slices were lightly counterstained with 0.05% cresyl violet for visualization of cytoarchitectural features.

Culture of Cortical Slices and Pulse-labeling with BrdU

E18–19 brains from littermates were removed and immersed in cold (4°C slicing medium (in mM: 120 NaCl; 5 KCl; 1.2 KH2PO4; 14 dextrose; 26 NaHCO3; 1.24 MgSO47H2O plus 5 mg/ml phenol red) and 300 µm coronal slices of the telencephalon were prepared using a vibratome. Slices were transferred onto millipore filters and placed into six-well Costar plates containing ice-cold slicing medium. The slices were allowed to recover for 2 h at 4°C and were then transferred on the filters to plates containing growth medium (Neurobasal medium supplemented with B27 and glutamine; Life Technologies, Grand Island, NY). At the beginning of the incubation period, the growth medium was supplemented with 50 µg/ml BrdU. After 18 h, BrdU-containing medium was replaced with growth medium lacking BrdU. To control for anatomical gradients of cortical maturation, as well as possible differences in cortical development among embryos of the same litter, slices from one cortical hemisphere cultured under control conditions were compared to slices of the contralateral hemisphere cut from the same embryo and maintained in media containing antagonist(s) (100 µM saclofen, 100 µM picrotoxin, a cocktail containing 100 µM picrotoxin plus 100 µM saclofen, or 100 µM bicuculline only). Antagonists were added at high micromolar levels to ensure that the ligands penetrated through the thick sections. Slices were cultured at 37°C in a mixture of 10% CO2 and 90% air. Six day cultures were refed every 2 days.

Viability of Cells in the Cultured Slices

The viability of cells in the slice cultures was monitored using propidium iodide (PI), which intercalates into double-stranded DNA and fluoresces a brilliant orange using the FITC filters. In some sets of studies, cultured slices were transferred from the filters onto glass microscope slides, and were completely covered with a drop of PI (50 µg/ml; Sigma, St Louis, MO). After a 5 min incubation at room temperature, the sections were coverslipped and examined immediately on a Zeiss Axiophot microscope, equipped with epifluorescence and the appropriate filters. Slices cultured for 3 days and then labeled with PI had very few cells with labeled nuclei; however, a few PI+ cells were observed scattered along the outermost edges of the slices. No difference in cell viability was noted between the antagonist-treated and the control slices. To ensure that the PI was able to penetrate through the slice, one set of slice cultures was kept in ambient air for 4 h at room temperature and then exposed to PI. The slices incubated at room temperature to induce cell death had an abundance of PI+ cells distributed throughout the cerebral wall.

To identify apoptotic cells in the slice cultures, TUNEL was performed using an ONCOR in situ peroxidase TUNEL kit (Intergen Co., Purchase, NY). Briefly, slices were cultured overnight in growth medium (see above). After 18 h, the medium was changed to either growth medium alone, or medium containing 100 µM saclofen, picrotoxin or a combination of the two antagonists. Slices were cultured in control or antagonist-containing medium for an additional 48 h, and were fixed for 1 h in 4% PF. Slices were rinsed three times in PBS, post-fixed for 5 min in ethanol/acetic acid (2:1), then washed twice in PBS. Endogenous peroxidase was quenched by incubating the slices for 5 min in 3.0% H2O2. Slices were exposed for 10 s to equilibration buffer, then incubated for 1 h at 37°C in TdT enzyme. Stop buffer was overlayed onto the slices for 10 min at room temperature. The slices were washed three times in PBS, incubated for 30 min at room temperature in anti-digoxigenin conjugate, and rinsed four times in PBS. Labeling was visualized using DAB. Following TUNEL, slices were briefly (30 s) counterstained with 0.05% cresyl violet for visualization of cytoarchitectural features. Stained sections were transferred onto glass slides and coverslipped. Slices were examined on a Zeiss Axiophot microscope using 5x, 10x, 16x or 25x objectives, and videomicroscopic images were captured using a CCD camera.

A few cells labeled by TUNEL were observed in all slices. The majority of labeled cells were observed scattered along the outermost edges of the slices at the pial surface (Fig. 1A,BGo) and at the vz, adjacent to the lateral ventricle (Fig. 1C,DGo); however, a few labeled cells were observed within all cortical regions including the cp (Fig. 1AGo), the iz (Fig. 1BGo), the vz and the svz (Fig. 1C,DGo), demonstrating that the technique labeled cells throughout the thick slices. The antagonist-treated and control slices exhibited no difference in either the number of cells labeled by TUNEL or the anatomical distribution of labeled cells. The majority of cells in all regions of the cultured slices were unlabeled, indicating that most cells in the slice cultures were not undergoing apoptosis.



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Figure 1. Identification of apoptotic cells in cultured slices by TUNEL. Videomicroscopic images of slices sectioned at E18, cultured overnight in Neurobasal medium plus B27, then cultured for an additional 2 days in Neurobasal/B27 medium with or without 100 µM saclofen or a combination of 100 µM saclofen plus 100 µM picrotoxin, then analyzed by TUNEL. Cells labeled by TUNEL are scattered along the outermost edge of the cp in the control (A) and antagonist-treated slice (B) (white arrows). A few positive cells are evident within the cp (A) and iz (B) (black arrows). Cells labeled by TUNEL are distributed at the innermost edge of the vz in both the control (C) and the antagonist-treated slice (D) (white arrows). A few positive cells are also evident within the svz (black arrows). Dashed lines (A,B) delineate the border between the cp and iz. cp, cortical plate; iz, intermediate zone; LV, lateral ventricle; svz; subventricular zone; vz, ventricular zone. Bar = 40µm.

 
BrdU Immunolabeling

Two or six days after removing BrdU from the cultures, the slices used for migration studies were fixed for 1 h in 4% PF, then permeabilized for 1 h in 0.5% Triton X-100. After rinsing in PBS, the slices were first incubated for 10 min in cold (4°C) 0.1 N HCl, then incubated for 25 min at 37°C in 1 N HCl. The HCl was removed and the pH of the slices was neutralized by adding 0.1 M Tris–HCl buffer, pH 8.0. The slices were then rinsed twice in PBS and incubated overnight at 4°C in the anti-BrdU antibody (diluted 1:33 in PBS) (Boehringer Mannheim, Indianapolis, IN). Immunolabeling for BrdU was visualized using the Elite Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and DAB. Following immuno- staining, slices were briefly (30 s) counterstained with 0.05% cresyl violet for visualization of cytoarchitectural features. Stained sections were transferred onto glass slides and coverslipped.

Slices were examined on a Zeiss Axiophot microscope using 5x, 10x, 16x or 25x objectives, and videomicroscopic images were captured using a CCD camera. NIH Image Analysis software was used to measure the density of BrdU-labeled nuclei and cortical plate diameter (thickness). Control and experimental data were taken from corresponding anatomical positions, and each videomicroscopic capture was exposed to the same analysis subroutine and density slice thresholding. Image analysis data was analyzed statistically using Student's t-test or ANOVA.

Density of BrdU-labeled Cells by Cortical Region

BrdU is incorporated into proliferating cells during DNA synthesis or the S-phase of the cell cycle. Therefore, it remains permanently integrated in the DNA and can be detected at any point during a cell's lifespan. If a cell incorporates BrdU during its terminal mitosis, all of the BrdU remains within that cell's DNA, and the subsequent immunostaining for the marker appears as a dense dark-brown label in the nucleus. However, when a cell that has incorporated BrdU during a pulse undergoes further proliferation, the BrdU becomes distributed among the nucleic DNA of the daughter cells, thus the BrdU immunoreaction signal is ‘diluted’. In these cells, the immunostaining appears pale in comparison to the cell incorporating BrdU during terminal mitosis. In these studies, only dense, dark-brown nuclei, which were interpreted as representing cells that underwent terminal mitosis during the BrdU-pulse, were considered positive and analyzed by densitometry (see Fig. 5Go). Cresyl violet staining allowed for the identification of the ventricular zone/subventricular zone (vz/svz), intermediate zone (iz) and cortical plate (cp) compartments (see Fig. 6AGo). The pixel density in each cortical compartment was measured, and the compartmental contribution to the total pixel density for the slice (% of total BrdU+ pixel density by region) was calculated. A total of 30 measurements on six coronal slices from three different littermates were collected and averaged for each trial. Illustrations present the averages (± SEM) from five trials.



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Figure 5. Densitometry of BrdU+ nuclei in anatomical compartments using image analysis. (A) A videomicroscopic grayscale image was captured. Only black nuclei were considered as representing cells that underwent terminal mitosis during the in vitro pulse, and were included in the counted population (blue arrows A,B). Pale-labeled nuclei (pink arrows A,B) were not included in the counted population. (B) The image was adjusted so that only black nuclei were selected for quantitation (overlayed in red). A polygon was drawn (yellow) to delineate the boundaries of the anatomical compartment to be measured (in this case, the cp), and the red pixel density within the polygon was measured. cp, cortical plate; iz, intermediate zone. Dashed lines delineate the boundaries between the iz and cp. Bar = 40 µm.

 


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Figure 6. Picrotoxin and/or saclofen treatment for 48 h affects the anatomical distribution of BrdU+ cells in cultured cortical slices. (A) Cresyl violet staining of a slice cultured for 3 days. The dashed lines delineate three anatomical compartments: the ventricular/subventricular zone (vz/svz), the intermediate zone (iz) and the cortical plate (cp). Densitometry of BrdU-immunoreaction signals was measured in each compartment (see Materials and Methods). Summary of quantitative densitometry in the three compartments under control conditions and in the presence of picrotoxin (pic) and/or saclofen (sac). In control slices (B), the majority of signals reflecting migrated cells are found in the cp. In slices treated for 48 h with a combination of 100 µM picrotoxin and 100 µM saclofen (C), saclofen only (D) or picrotoxin only (E), the majority of labeled cells are located in the ventricular zone/subventricular zone (vz/svz) and intermediate zone (iz). Bar (A) = 80 µm. Error bars = SEM. Values are means from five separate trials. *P <= 0.05, Student's t-test.

 
Statistical analysis of the pixel density per compartment data was initially assessed using a two-factor ANOVA, which confirmed that there was a significant interaction between compartment and culture condition. Single-factor ANOVAs were then used to assess differences in pixel density for each individual compartment among the experimental culture conditions. To identify where the significance existed (P <= 0.05), the ANOVA analyses were followed by Scheffé's S post hoc tests.

Cortical Plate Thickness

Using the NIH Image software, the linear distance (in pixels) was measured from the innermost edge of cortical layer VI to the outermost edge of cortical layer I (see Fig. 7Go). This measurement was assessed in four places in each slice, and averaged. Cortical thickness in control slices was compared to the thickness in contralateral treated slices. Significance between experimental and control cp thickness was assessed using Student's t-test.



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Figure 7. Bicuculline alters the anatomical distribution of BrdU+ cells in cultured cortical slices. Videomicrographs of cortical slices cultured in either control medium (A), or medium containing 100 µM bicuculline (B). Arrows (A,B) indicate the extent of the cp. Dashed line delineates the border of the cp and iz. (C) Alignment of the arrows used to measure cp thickness in (A,B). The cp thickness is larger in the bicuculline-treated slice (bic) than in the control (cont). Arrowheads (A,B) indicate examples of BrdU+ nuclei. cp, cortical plate; iz, intermediate zone. Bar = 40 µm.

 
Immunostaining Cortical Slices for Nestin or GFAP

E18 or E21 acute vibratome sections or the cultured slices (slices cut on E18 and maintained in vitro for a total of 3 days) were fixed for 1 h in 4% PF, rinsed, then incubated for 48 h at 4°C in rabbit anti-nestin antibody (1:1500; gift of Dr R. McKay, NINDS, NIH, Bethesda, MD) or in rabbit anti-GFAP (1:500; Sigma). PBS with 0.05% bovine serum albumin (BSA) and 0.01% saponin was used as the primary antibody diluent. The sections were rinsed in PBS and the antibody staining was visualized using the Elite Vectastain ABC kit and DAB. Following the immuno- labeling, the sections were briefly counterstained with 0.05% cresyl violet for visualization of cytoarchitectural features. Stained sections were transferred onto glass slides and coverslipped.


    Results
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Glial Cell Maturation in Acute and Cultured Slices

To evaluate the maturation of glial cells in the cultured slices, sections were immunostained with antibodies directed against nestin, an intermediate filament protein expressed by precursor cells and radial glia (Tohyama et al., 1992Go). At the time of plating, acutely harvested E18 sections contained nestin+ elements that displayed a radial morphology throughout the cp (not shown). An examination of sections harvested acutely at E21, however, demonstrated that nestin+ cell processes in the cp had a branched morphology (Fig. 2AGo), suggesting that radial glia were transforming into astrocytes. Slices that were harvested on E18 and cultured for 3 days (3 Div, equivalent to E21 in vivo) showed a pattern of nestin immunoreactivity that paralleled the pattern observed in slices acutely prepared at E21 (Fig. 2BGo). Immuno- staining sister slice cultures for the astrocyte marker, glial fibrillary acidic protein (GFAP), revealed that by 3 days in vitro, some cp cells in the cultured slices expressed GFAP (Fig. 2DGo). Immunostaining acutely harvested sections at E21 confirmed that GFAP was detectable in some cp cells (Fig. 2CGo). Thus, astroglial maturation in the cultured slices paralleled the differentiation of astrocytes in vivo.



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Figure 2. Nestin and GFAP immunoreactivity in cortical slices demonstrates that glial cell maturation is similar in vitro and in vivo. Videomicroscopic images show acute (A) and cultured slices (B) that have been immunostained with anti-nestin antibody. (A) A cortical slice harvested on E21, fixed and stained with anti-nestin antibody, demonstrates nestin-immunoreactive fibers in the cp which exhibit a branched morphology (arrows). (B) A cortical slice sectioned at E18, then cultured for 3 days (3 Div), shows a pattern of nestin immunoreactivity similar to that observed in the slice processed at E21. (C) A slice harvested on E21 shows GFAP+ cells distributed in the cp (arrows). (D) A slice from E18 that was cultured for 3 days (3 Div) has GFAP+ cells in the cp (arrows). cp, cortical plate. Bar = 30 µm.

 
Many Cells Labeled with BrdU at E18 In Vivo and In Vitro Migrate from the VZ into the CP within 3 Days

Cortical slices harvested on E18, 4 h after an in vivo injection of BrdU, had cells with BrdU+ nuclei widely distributed throughout the vz (Fig. 3AGo). No BrdU+ cells were apparent in the cp of the E18 slices. In contrast, sections harvested 3 days after the in vivo injection, on E21, had many BrdU-labeled cells in the cp (Fig. 3CGo). Immunopositive cells were rarely observed in the vz of the E21 slices (not shown). These results demonstrate that many vz cells that incorporated BrdU in vivo at E18 had migrated out of the germinal regions and entered the cp within 3 days.



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Figure 3.  In vivo and in vitro, most BrdU+ cells migrate from the vz to the cp within 3 days. Videomicroscopic images of sections pulsed with BrdU and immunostained to show the localization of BrdU+ cells in the cortex. (A) An E18 cortical section harvested 4 h after an in vivo BrdU pulse shows many cells with BrdU+ nuclei (black dots, arrows) confined to the vz. (B) A section cultured on E18 and pulsed overnight in vitro also has many BrdU+ cells in the vz (arrows). (C) A section harvested on E21, 3 days after an in vivo BrdU pulse (at E18), reveals many BrdU-labeled cells in the cp (arrows, C). (D) A slice cultured on E18, pulsed overnight with BrdU in vitro, and immunostained for BrdU 48 h later (3 Div) has many BrdU+ cells in the iz and cp (arrows, D). cp, cortical plate; iz, intermediate zone; LV, lateral ventricle; vz, ventricular zone. Dashed lines (C,D) delineate the border of the cp and intermediate zone. Bar = 40µm.

 
The temporal pattern of postmitotic vz cell migration in cultured cortical slices was similar to the pattern observed in vivo. Cortical slices harvested on E18 and cultured for 18 h in BrdU had immunopositive cells throughout the vz (Fig. 3BGo). Slices that were pulsed in vitro, then cultured for an additional 2 days in BrdU-free medium (for a total of 3 days in vitro) had BrdU+ cells throughout the iz and cp (Fig. 3DGo). Thus, under these experimental conditions, many vz cells that incorporated BrdU in vitro during the initial 18 h also migrated out of the germinal zones and entered the cp within 3 days. These results demonstrate that temporal patterns of neuronal migration are similar in vivo and in vitro.

GABA Receptor Antagonists Retard BrdU-labeled Cells from Migrating into the CP in Cultured Slices

Prior motility studies on dissociated cortical cells showed that GABA stimulates cell movement in distinct subsets of embryonic cells via multiple classes of GABA-Rs (Behar et al., 1996Go, 1998Go). In those studies, a combination of saclofen (a GABAB-R antagonist) (Kerr and Ong, 1992Go) and picrotoxin (a GABAA/GABAC-R antagonist) (Jackel et al., 1994Go) completely blocked GABA- induced chemotaxis of vz cells (Behar et al., 1996Go). In vivo, if endogenous GABA released by cortical cells directs vz cells to migrate towards the cp, then a combination of saclofen and picrotoxin should inhibit cell migration in the cultured slices.

Therefore, we examined the effect of the two antagonists on vz cell migration in slice cultures. Following overnight exposure to BrdU to label premigratory cells, slices were maintained for 48 h in either BrdU-free medium, or medium containing saclofen and picrotoxin. Treatment with the GABA-R antagonists altered the distribution of BrdU-labeled cells within the slices. Slices maintained in medium containing a mixture of the two antagonists had an abundance of BrdU+ cells in the vz (Fig. 4DGo). In contrast, few labeled cells were observed in the vz of the matched, control slices (Fig. 4CGo). While the control slices had an abundance of labeled cells in the cp (Fig. 4AGo), there were few labeled cells in the cp of the treated slices (Fig. 4BGo). These results suggested that in antagonist-treated slices, the vz cells which had incorporated BrdU remained within the germinal regions and failed to migrate into the cp.



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Figure 4. A combination of picrotoxin and saclofen blocks BrdU+ cell migration in slices. Videomicroscopic images of sections harvested at E18, then pulsed with BrdU for 18 h and cultured an additional 2 days in Neurobasal/B27 medium with or without a mixture of 100 µM picrotoxin and 100 µM saclofen. (A) The slice cultured in control medium has an abundance of BrdU-labeled cells in the cp (arrows). (B) Few BrdU+ cells have entered the cp of the contralateral slice treated with picrotoxin and saclofen (arrows). (C) A slice cultured in control medium has few labeled vz cells (arrows) while the vz of the contralateral slice cultured with picrotoxin and saclofen has many cells with BrdU+ nuclei (arrows) (D). Dashed lines (A,B) delineate the innermost aspects of the cortical plates in the sections. cp, cortical plate; iz, intermediate zone; LV, lateral ventricle; vz, ventricular zone. Bar = 40 µm.

 
To determine if exposure to the antagonists led to a decrease in the BrdU+ population, densitometry was used to quantitate BrdU-labeled nuclei in the cultured slices (see Materials and Methods; Fig. 5Go). Forty-eight hours after removing the medium with BrdU, the total pixel density in control slices did not differ significantly from the total pixel density in the treated slices (P <= 0.05), indicating that treatment with the two antagonists did not result in the elimination of BrdU+ cells. Slices maintained in control medium had an average total pixel density of 23 703 (±3404), whereas slices maintained in a mixture of picrotoxin and saclofen had an average pixel density of 19 615 (±3997). These data indicate that there was no significant difference in the number of BrdU+ nuclei in the treated versus control slices, indicating that exposure to the antagonists did not result in an overall depletion of BrdU+ cells, but rather a redistribution of the cells throughout the cortical anatomy.

To confirm that antagonist exposure resulted in an altered distribution, image analysis and densitometry were used to quantitate the relative number of BrdU+ cells in each of three cortical compartments: the ventricular/subventricular zone (vz/svz), the intermediate zone (iz) and the cortical plate/subplate (cp). These compartments were readily discernible in cultured slices that were counterstained with cresyl violet (Fig. 6AGo). For densitometry, a videomicroscopic greyscale image of a slice was captured (Fig. 5AGo). For quantitative purposes, only black nuclei were considered as representing cells that underwent terminal mitosis during the in vitro pulse period, and were included in the counted population. The image was adjusted so that only the black nuclei were selected for quantitation (Fig. 5BGo, red), and a polygon was drawn to delineate the anatomical area of interest (in this case the cp, Fig. 5BGo). The pixel density of the selected (red) regions within the polygon was measured, and the contribution of a compartment's pixel density to the total pixel density of the slice was calculated (% total pixel density). Densitometry confirmed that 48 h after removing BrdU, the majority of labeled cells in the control slices were located in the cp (76.3 ± 5.1%) (Fig. 6BGo). In contrast, slices treated for 48 h with either picrotoxin, saclofen or a combination of both antagonists had a small proportion of BrdU+ cells in the cp (24.7 ± 16.2%; 13.4 ± 4.8% and 25 ± 6.3% respectively) (Fig. 6CEGo). Most of the BrdU+ cells in the antagonist-treated slices were located in the vz/svz and iz — i.e. cortical compartments associated with immature cells.

Saclofen and picrotoxin appeared to have different effects on cell migration in the cultured slices. Whereas saclofen prominently inhibited the movement of cells out of the iz, picrotoxin predominantly blocked the movement of cells out of germinal regions. Slices treated with saclofen alone had most BrdU+ cells in the iz (64.0 ± 2.3%, Table 1Go). In contrast, almost half of all labeled cells remained within the vz/svz of slices exposed to picrotoxin (45.0 ± 15.1%, Table 1Go).


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Table 1 GABA receptor antagonists alter the anatomical distribution of BrdU+ cells in cultured cortical slices
 
After 6 days of culture, the distribution of BrdU+ cells had changed in the control and treated slices (Table 1Go). In controls and in the picrotoxin-treated slices, virtually all (98%) of the BrdU+ cells were found in the cp (Table 1Go). In contrast, only 73.2% (±2.8%) of BrdU+ cells were found in the cp of the saclofen-treated slices. When saclofen was present, one-quarter of all BrdU+ (25%) cells were still evident in the iz after 6 days; however, the vz was virtually devoid of labeled cells (Table 1Go). Slices treated with a combination of saclofen and picrotoxin showed an anatomical distribution of BrdU+ cells that was similar to the distribution observed when slices were exposed to saclofen only (Table 1Go). These results indicate that blocking GABA-Rs with picrotoxin or saclofen delayed, but did not completely arrest, the migration of BrdU+ vz cells into the cp. The effects of these two antagonists on the cultured slices complement the observations on GABA-induced migration of dissociated cortical cells, and suggest that saclofen- and picrotoxin-sensitive GABA-Rs mediate the pro-migratory signals of endogenous GABA.

Bicuculline, a GABAA-R Antagonist, Does Not Block Migration of BrdU+ Cells into the CP

Picrotoxin is an antagonist of both GABAA and GABAC receptors. To determine if the observed effects of picrotoxin involved GABAA-R activation, we evaluated the effect of bicuculline, a specific GABAA-R antagonist (Sivilotti and Nistri, 1991Go), on vz cell migration in the cultured slices. In the present study, bicuculline failed to significantly retard the movement of BrdU+ cells into the cp (Table 1Go, Figs 7, 8A,BGoGo), indicating that the pro-migratory effects of GABA involved picrotoxin-sensitive, bicuculline-resistant receptors. Interestingly, slices treated for 48 h with bicuculline had an increase (of ~30%) in the relative number of BrdU+ cells in the cp (Table 1Go), implying that GABAA-R activation may provide a ‘stop’ signal for migrating cells.



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Figure 8. Pharmacologically induced changes in cortical plate thickness parallel induced-changes in the number of BrdU+ cells in the cortical plate. Histogram illustrates the relative density of BrdU-labeled cells (black bars) and the extent of the cp (stippled bars) in antagonist-treated slices. In each condition, a change in the cp thickness parallels a similar change in the relative density of BrdU-labeled cp cells. Treatment with saclofen, picrotoxin or a combination of both antagonists leads to a decrease in BrdU+ cp cells and a decrease in cp thickness compared to controls. In contrast, bicuculline treatment enhances migration of BrdU+ cells into the cp, and results in increased cp thickness. *P <= 0.05, Student's t-test.

 
Cortical Plate Thickness Decreases in Picrotoxin- and Saclofen-treated Slices, but Increases when Slices are Exposed to Bicuculline

Since there was an increase in the relative number of BrdU+ cp cells following bicuculline treatment, the thicknesses of cortical plates were measured (from the innermost edge of layer VI through outermost edge of layer I) in the treated and untreated cultured slices (Fig. 7Go and see Materials and Methods). Not surprisingly, the average thickness of the cp was increased in slices that were maintained for 2 days in bicuculline (138 ± 18.0% of control, Figs 7, 8GoGo). However, the slices maintained for 2 days in saclofen, picrotoxin or a mixture of both antagonists showed decreases in cp thickness (by 52.1 ± 9.9%, 37.9 ± 28.9% and 26.6 ± 27.0% respectively) (Fig. 8Go). Thus, 48 h after the BrdU pulse, changes in cp thickness paralleled changes in the relative number of BrdU+ cp cells. When the the number of BrdU+ cp cells was decreased, the cp thickness was diminished. Conversely, an increased density of BrdU+ cells in the cp corresponded to increased cp thickness (Fig. 8Go).


    Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Slice Culture Strategy Preserves Migration

We have used stationary slice cultures of the developing cerebral cortex to study receptors that modulate embryonic postmitotic cell migration. Slice cultures of nervous system tissue have been used extensively to study developmental processes such as cell growth and differentiation (del Rio et al., 1996Go, 1997Go; Lavdas et al., 1997Go; Arimatsu and Ishida, 1998Go; Vogt et al., 1998Go; Klostermann and Wahle, 1999Go), innervation (Baratta et al., 1996Go; Dantzker and Callaway, 1998Go; Kluge et al., 1998Go), cell migration (Caeser et al., 1989Go; Roberts et al., 1993Go; Kunimoto and Suzuki, 1997Go; Hartmann et al., 1998Go; Ohnishi et al., 1998Go), as well as pathological conditions such as neurotoxicity and neuro degeneration (Yoshikawa et al., 1998Go; Noraberg et al., 1999Go; Teter et al., 1999Go). Under appropriate conditions, slice cultures provide adequate access to developmentally relevant events occurring in dynamically changing tissue, in which the cyto-architecture is preserved, cell–cell contacts remain intact and many physiological properties are maintained.

The preservation of cell–cell interactions is an important feature of slice cultures since, in vivo, migration of many cortical neurons appears to involve direct contact between neurons and radial glia. As postmitotic neurons migrate away from the vz, many neurons are thought to migrate along the processes of radial glial cells, which serve as a scaffold to guide neurons towards their target positions in the cp (Hatten, 1990Go; Rakic, 1990Go). Near birth, radial glia reportedly begin to transform into astrocytes (Hunter and Hatten, 1995Go). Hence, the initial preservation and eventual maturation of radial glia are important characteristics if cultured slices are to serve as models for studying mechanisms operable in vivo. Although Götz and Bolz (Götz and Bolz, 1992Go) reported that astrocytes rarely developed in their cultures of embryonic cortical slices, under the conditions used in the present study, immunohistochemistry confirmed that the onset of astrocytic differentiation in the cultured slices occurred on schedule, and paralleled the emergence of cortical astrocytes observed in vivo (Hunter and Hatten, 1995Go).

To verify that temporal patterns of neuronal migration in cultured slices mimicked patterns of cortical neuronal migration in vivo, proliferating cells were pulsed with BrdU between E18 and E19. Forty-eight hours later, the distribution of BrdU+ cells was observed in vivo and in vitro. Proliferating neuroblasts incorporate BrdU while in the vz, and thus the distribution of BrdU+ cells outside of the germinal regions was taken as an indicator of postmitotic vz cell migration. Both in vivo and in vitro, many BrdU+ cells that underwent terminal mitosis between E18 and E19 were found to have entered the cp by E21 (or 3 Div). Thus, temporal patterns of postmitotic vz cell migration were similar in the organotypic cultures and in vivo, verifying that short-term cultured slices are appropriate models for studying embryonic neuronal migration. In vivo, most cells born between E18 and E19 are neurons destined to form cortical layers II and III (Bayer, 1990Go; Jacobson, 1991Go), suggesting that the densely labeled BrdU+ cells in the cultured slices were neurons destined for these two lamina.

GABA Receptor Antagonists Reveal GABA's Chemoattractant Roles In Situ

Previous in vitro studies using acutely prepared cortical cell dissociates demonstrated that GABA can act as a chemoattractant, stimulating embryonic nerve cell movement (Behar et al., 1996Go, 1998Go). In those studies, femtomolar concentrations of GABA induced chemotaxis of neurons dissociated from immature cortical regions (vz/svz and lower iz) (Behar et al., 1998Go). Pharmacological characterization of the migratory response revealed that saclofen- and/or picrotoxin-sensitive receptors mediated pro-migratory signals (Behar et al., 1996Go). The results using slice cultures support the observations on the pharmacology of GABA-induced migration observed with dissociates, and place these findings within an anatomical context. Here, a 48 h saclofen and picrotoxin treatment prevented most BrdU+ cells from entering the cp, providing evidence that GABA directs cells to migrate into this anatomical compartment.

Interestingly, a 2 day saclofen exposure only mildly retarded the movement of cells out of the germinal regions; in the presence of saclofen, >60% of the BrdU+ cells in the slices were localized in the iz, indicating that inhibition of GABAB-Rs did not prevent most cells from leaving the proliferative zone and beginning their migration towards the cp. In contrast, a 2 day picrotoxin exposure appeared to have a far more pronounced effect, retarding the migration of vz/svz cells. A significant proportion of all BrdU+ cells remained within the germinal regions of slices cultured for 48 h in picrotoxin. In the presence of picrotoxin, there was approximately a 2-fold increase in the number of labeled cells localized in the vz/svz compared to saclofen-treated slices, and a 9-fold increase over the proportion found in the vz/svz of control slices. These results suggest that picrotoxin-sensitive GABA receptor activation is involved in signalling cells to leave the vz/svz and enter the iz, while saclofen-sensitive receptor activation directs cells to leave the iz and enter the cp. Thus, two pharmacologically distinct classes of GABA-Rs may be involved in modulating neuronal movement along migratory routes.

In the mature central nervous system, three classes of GABA-Rs have been described. GABAB-Rs are metabotropic receptors that couple to G-proteins (Kerr and Ong, 1992Go), whereas GABAA- and GABAC-Rs form Cl channels (Sivilotti and Nistri, 1991Go; Feigenspan et al., 1993Go). Saclofen is a specific antagonist of GABAB-Rs; hence, the inhibitory effects of saclofen observed in the present studies directly implicate GABAB-R activation in promoting nerve cell movement. Picrotoxin, however, blocks both GABAA- and GABAC-Rs, while bicuculline is a selective GABAA-R antagonist (Feigenspan et al., 1993Go). In these studies, only picrotoxin blocked the migration of BrdU+ cells; bicuculline failed to inhibit migration, indicating that pro-migratory signals of GABA involve the activation of picrotoxin-sensitive, bicuculline-resistant, GABAC-like receptors. Alternatively, in the embryonic cortex, migrating neurons may express novel G-protein coupled GABA-Rs that are picrotoxin sensitive. In support of this hypothesis, the low, femtomolar concentrations of GABA that promote acutely dissociated vz cells to migrate in vitro would not be expected to activate ionotropic GABAA and/or GABAC receptor/Cl channels. In addition, pertussis toxin, a selective blocker of Gi and Go proteins (Ui, 1984Go), completely blocked GABA-induced migration of dissociated E18 vz cells in a modified Boyden chamber assay (Behar et al., 1998Go). Together, these findings suggest that vz/svz cells may express unique picrotoxin-sensitive GABA-Rs that couple to G-proteins.

GABAA Receptors Mediate Arrest of Migration In Situ

Whereas activation of saclofen- and picrotoxin-sensitive receptors promotes migration, activation of GABAA-Rs may actually arrest cell movement. Blockade of GABAA-Rs with bicuculline resulted in a statistically significant increase in the relative number of BrdU+ cells in the cp, as well as an increase in cp thickness, suggesting that GABAA-R activation signals cells to stop moving. The GABAA-R subunits expressed by embryonic cells in the cp differ from the subunits expressed by cells in more immature regions of the cortex (Ma and Barker, 1995Go). In vivo, activation of GABAA-Rs expressed by cp cells may provide a ‘stop’ signal for migrating neurons as they approach their target destinations. Evidence that GABAA-Rs mediate ‘stop’ motility signals in the slices corroborates observations from prior in vitro studies on cortical dissociates, where cp cell migration induced by GABAmimetics was potentiated in the presence of bicuculline, and blocked in the presence of the GABAA-R agonist, muscimol (Behar et al., 1998Go).

The results of the slice studies indicate that multiple classes of GABA-Rs modulate the movement of migrating embryonic cortical neurons. The intracellular signalling mechanisms that promote or arrest cell movement remain to be resolved; however, they are likely to involve modulation of intracellular Ca2+ levels. In dissociated cells, GABA-induced migration is dependent upon increased cytosolic Ca2+ ([Ca2+]c) (Behar et al., 1996Go). However, low and high GABA concentrations elicit distinct patterns of increased [Ca2+]c in cell dissociates. Studies on granule cell migration and growth cone turning revealed that distinct patterns of Ca2+ fluctuations either promote or retard cell movement (Gomez et al., 1995Go; Horgan and Copenhaver, 1998Go; Komuro and Rakic, 1998Go; Gomez and Spitzer, 1999Go). While the Ca2+ response to femtomolar GABA may be permissive for cell movement, the [Ca2+]c response to high GABA levels (and GABAA-R activation) may inhibit motility.

Analysis of the distribution of BrdU-labeled cells in slices maintained in antagonists for 48 h and 6 days revealed that exposure to saclofen or picrotoxin resulted in a delay, but not a complete arrest, of vz cell migration into the cp. These findings suggest that the motility-promoting effects of GABA-R activation may regulate the rate of neuronal migration in the developing cortex. Amino acids have been shown to regulate the rate of neuronal motility through Ca2+-dependent mechanisms in the developing cerebellum. Komuro and Rakic reported that glutamate influenced the rate of granule cell migration in postnatal cerebellar slices by modulating levels of [Ca2+]c (Komuro and Rakic, 1998Go). GABA may serve a similar role during cortical morphogenesis.

During the final week of gestation, GABA is transiently expressed in several regions of the cortex. At this stage of development, GABAergic cells are found in layer I, the cp and the subplate (Imamoto et al., 1994Go; Behar et al., 1996Go). GABA released from cells in these regions could provide chemotropic stimuli to postmitotic neurons, directing the neurons to migrate out of the germinal zones, through the iz, and into the cp. In the present study, GABA-R blockade prevented cells from migrating into the cp; however, GABA was not added to the culture medium. These findings suggest that the cortical cells in the slices synthesized and released GABA, which in turn, influenced the movement of newly generated neurons.

In summary, the pharmacological studies on cultured cortical slices indicate that GABA, released by cortical cells, stimulates postmitotic neurons destined for cortical layers II and III to migrate into the cp via activation of saclofen- and picrotoxin- sensitive receptors. While picrotoxin-sensitive receptors appear to prompt cells to move out of germinal regions, saclofen- sensitive receptors may direct cells to migrate into the cp. The results of the bicuculline studies on the cultured slices indicate that GABAA-R activation arrests embryonic cortical cell movement (Fig. 9Go). All of these findings corroborate the results from earlier studies on cell dissociates, and provide evidence that in the developing rat cortex, GABA acts as a chemoattractant modulating cell migration during cortical histogenesis.



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Figure 9. GABA-Rs modulate the movement of embryonic neurons along migratory routes. During development, GABA is expressed near the target destinations for migrating neurons. GABA released from cells in these regions may diffuse through the tissue, signalling newly generated neurons in the ventricular zone to migrate. Picrotoxin-sensitive receptor activation signals cells in germinal zones to leave the vz/svz and enter the iz, while GABAB-R activation directs cells in the iz to enter the cp. Once in the cp, the neurons acquire GABAA-R subunits that, when activated by high concentrations of GABA, arrest migration as the cells approach their target destinations. Thus, changing receptor expression may regulate motility responses to GABA.

 

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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Address correspondence to T.N. Behar, Building 36, Room 2C02, 36 Convent Drive, Bethesda, MD 20892–4066, USA. Email: behart{at}ninds.nih.gov


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