A model of cortical dysplasia results from disruption of the earliest generated neocortical cells. Injections of an antimitotic (methylazoxy methanol — MAM) into pregnant ferrets result in a constellation of effects, which include disruption of radial glia, with early differentiation in astrocytes, and impaired migration of neurons into the cortical plate. We found previously that culture of P0 MAM-treated slices with explants of normal cortical plate reorganizes the radial glia toward their normal morphology and improves migration of neurons into the cortical plate. This suggested that P0 normal cortical plate contains a ‘factor’ capable of providing reorganizing cues to disorganized developing cortex. The current study characterizes the biological activity in normal cortical plate by isolating fractions of different molecular weight obtained from conditioned media of organotypic cultures. The only media fraction capable of providing reorganizing activity to MAM-treated cortex was the molecular weight fraction between 30 and 50 kDa. Treatment designed to denature proteins demonstrated that the active molecular weight fraction (30–50 kDa) was not able to provide reorganizing cues when either heated or treated with Proteinase K. These data provide support for the idea that normal cortical plate of neonatal ferret contains a radialization factor that is a protein of 30–50 kDa.
Deficits in neocortical migration can result from numerous factors. We developed a model of cortical dysplasia that results in a constellation of effects. By injecting an antimitotic into pregnant ferrets during early phases of corticogenesis, a precise pattern of disruption occurs in the brains of treated animals. These include: evidence of a thin cortical plate with disrupted lamination, disorganized radial glial cells with precocious differentiation into astrocytes, and Cajal Retzius cells that are disordered and no longer located primarily in the marginal zone (Noctor et al., 1999). Because injections of this antimitotic (methylazoxy methanol — MAM) specifically interrupt the birth of cells that would normally populate the earliest generated cortical layers, we hypothesized that biological activity (a ‘factor’) generated by these cells was missing from the MAM-treated cortex, which resulted in the collection of abnormal effects. If this was so, we proposed that the missing ‘factor’ should be present in normal cortical plate.
To test this hypothesis, we co-cultured slices of MAM-treated ferret cortex with normal cortical plate. These studies demonstrated that normal cortical plate cultured adjacent to MAM-treated cortex re-established the shape of radial glial cells toward their normal elongated morphology, improved migration of neurons into the cortical plate, and restored Cajal Retzius cells to the marginal zone. Co-culture with MAM-treated cortex did not restore these features (Hasling et al., 2003).
The current studies further describe the biological activity present in normal cortical plate. We determine that the ‘factor’ contained in normal P0 cortical plate is a protein of between 30 and 50 kDa capable of restoring radial glia to their normal morphology and improving migration into the cortical plate.
Materials and Methods
This study characterizes the biological activity present in normal cortical plate employing conditioned medium from normal P0 cortical organotypic cultures. The conditioned medium is used either by itself, or after fractionating it into aliquots of different molecular weight (MW) and adhering the aliquots to fluorescent microspheres. The microspheres are then injected into organotypic cultures of E24 MAM-treated cerebral cortex and maintained for 2 days. The organotypic cultures are assessed by evaluating the ability of the microspheres with adhered conditioned medium to alter the morphology of radial glia, or to improve neuronal migration by assessing the distribution of newly born cells labeled with bromodeoxyuridine (BrdU).
Injections of MAM and BrdU
Timed pregnant ferrets obtained from Marshall Farms (New Rose, NY) were anesthetized with isofluorane (3%) and nitrous oxide (0.05%) and injected i.p. with methyl azoxymethanol acetate (MAM) at a dose of 12 mg/kg on embryonic day 24 (E24). The MAM was dissolved into 5 cc of saline. After injection, the anesthesia was withdrawn and the animal monitored carefully until alert. MAM is a short acting antimitotic that prevents the birth of cells over about an 8 h period in the animal model and dose used here. MAM methylates guanine nucleotides (at the second carbon) of single-stranded DNA (7′ end) during S-phase, which prevents DNA polymerase from replicating the DNA, the cell arrests in S-phase, and effectively halts mitosis (Evans and Jenkins, 1976).
The injections of BrdU were made into ferret kits on P0 (the day of birth) prior to preparation of the organotypic cultures. The BrdU (50 mg/kg) was dissolved in saline with 0.007 N NaOH and injected i.p. three times at 1 h intervals. Ten pregnant ferrets were used for these experiments (some of the kits were used for other studies). Seven of these received injections of MAM on E24. Animals that did not receive MAM injections were used for normal controls, or to generate conditioned media from normal organotypic cultures. A total of 97 organotypic cultures were used for analysis in the experiments presented here.
Preparation of Organotypic Cultures
P0 ferret kits that previously received an injection of MAM on E24, or normal kits, were anesthetized i.p. with 50 mg/kg Pentobarbital Na. When insensitive to pain, their brains were removed and sliced on a tissue chopper at 500 μm in thickness. The region of presumptive parietal cortex was chosen to place in culture. At this age (P0), parietal cortex can be identified by morphological features (Juliano et al., 1996; Noctor et al., 1999). Each slice was placed in a 70 μm nylon mesh cell strainer (Becton Dickinson-Falcon, Bedford, MA), which was then positioned in a six-well tissue culture plate (Becton Dickinson-Falcon). Medium was added to the wells and the level maintained at the upper surface of the slice (Stoppini et al., 1991). The slice cultures were maintained in an incubator (37°C, 95% air/5% CO2) for the duration of the culture period. We used serum-free medium composed of Neurobasal (Gibco, Carlsbad, CA), with B-27 and N-2 supplements (Gibco) with added gentamycin and l-glutamine. Each slice culture received an injection of fluorescent microspheres with conditioned media fractions adhered (see below) and remained in culture for 2 days. After this, each culture received an injection of fluorescent dextran to selectively mark a set of radial glial cells, as described below, and was then placed in fixative (4% paraformaldehyde in 0.1 M phosphate buffer) overnight or longer, stained with bisbenzimide and subsequently analyzed. We also used organotypic cultures from normal P0 ferret brains for controls.
Fractionation of Conditioned Media and Incubation with Fluorescent Microspheres
Organotypic cultures of parietal cortex were prepared from normal ferret kits on P0 as described above. After 2–3 days in culture, the medium was collected (an average 1.26 ml of medium was obtained from a single source slice). The medium was centrifuged while passing through a Centrifugal Filter Device (Amicon Centriprep YM, Millipore, Billerica, MA) with cut-off limits of 50, 30 and 10 kDa MW, consecutively. The fractions were collected and immediately frozen in aliquots equivalent to the medium obtained from a single cultured source slice.
To adhere the conditioned medium of different MWs to fluorescent latex microspheres IX (LumaFluor, Naples, FL), 10 μl of microsphere suspension were added to conditioned medium with no treatment, or to a conditioned media fraction; the microspheres were added to a volume equivalent to that of one cultured slice. The microspheres were incubated for 30 min in the dark at RT on a shaker; they were then spun for 1 h using a bench centrifuge at maximum speed. The microspheres were then resuspended in 10 μl of fresh medium or sterile water and injected into each organotypic culture using a glass pipette with a tip size of 30–40 μm using a Picospritzer; each slice received an injection of ∼1 μl. Each injection was positioned in the center of the cortical plate. We were successful in the position of the injections, but occasionally the fluorescent microspheres were less specifically located.
Treatment to Deactivate Proteins
Two treatments were used to deactivate proteins. The first used thermal deactivation by heating the conditioned media, prior to addition of the microspheres, for 1 h at 68°C. The second employed enzymatic degradation of the conditioned media fractions using Proteinase K immobilized on a Matrix F7m-26-filled column (MoBiTec, Göttingen, Germany).
Injections of Fluorescent Dextrans
After being in culture for 2 days with injections of fluorescent microspheres, each organotypic culture was removed from the incubator and placed in a chamber used to maintain living slices as described previously (Juliano et al., 1996). Each culture received an iontophoretic injection of fluorescently labeled dextrans (4 μA, alternating positive current for 3 min), using glass pipettes with a tip bore of 20–30 μm. The injection sites were made in the intermediate zone adjacent to the dorsal and lateral portions of parietal cortex (somatosensory cortex) ∼100 μm into the thickness the slice. The slices were maintained in the chamber for 5–8 h at room temperature to allow for cellular uptake and transport of the dextran (Juliano et al., 1996). We previously observed that high MW dextrans are not taken up or transported in non-viable slices (we usually used Fluororuby; Molecular Probes, Eugene, OR) (Juliano et al., 1996; Noctor et al., 1999). The slices were then placed in 4% buffered paraformaldehyde for 12 h or longer. Before mounting on subbed slides, the slices were counterstained with bisbenzimide trihydrochloride (Sigma, St Louis, MO).
On P0, prior to culture preparation, the E24 MAM-treated kits received i.p. injections of BrdU as described above. Following the culture period, slices were removed and treated with 95% ETOH and 5% acetic acid for 30 min, 1 mg/ml pepsin (Sigma) in 2N HCl for 1 h at 37°C, and then rinsed in 0.1 M PBS pH 8.5 [adapted from Miller and Nowakowski (Miller and Nowakowski, 1988)]. Slices were incubated in mouse anti-BrdU (Boehringer-Mannheim, Indianapolis, IN) (1:20 in 0.1 M PBS pH 8.5) at 4°C overnight and washed in 0.1 M PBS pH 7.4; the slices were then incubated in the secondary antibody (Vectastain anti-Mouse Elite ABC kit, Vector Labs, Burlingame, CA) for 1 h. The BrdU label of the organotypic slices was visualized using a Vector VIP kit.
Analysis of Radial Glial Morphology
The label resulting from the dextran injections was quantified by measuring the angular divergence of labeled processes (Fig. 1). Each dextran injection was digitally imaged at ×25 to allow individual processes to be traced and converted to line drawings. In order to trace entire glial processes labeled by the dextran injections, several focal planes for each injection were imaged and consolidated. A representative line was created for each labeled process, starting from the injection site to termination of the process. The angle of each representative line was measured in degrees from a standardized line (representing 0°); the mean angle for the injection was then calculated. The absolute difference from the mean angle was calculated for each labeled process in the injection. The average of the absolute differences represented the mean angular deviation of each injection. The mean angular deviation was compared for statistically significant differences between conditions using the test of Least Significant Difference.
where α = angle of process from the standardized line representing 0°.
BrdU labeled cultures were digitally imaged at 25×; only the most densely labeled nuclei were considered for analysis. In each culture, the site of the fluorescent microspheres was determined and the number of BrdU positive cells counted in the vicinity of the injection and along the cortical plate in bins 300 μm in width that extended perpendicular from the pia to a depth of 100 μm (this includes the approximate depth of the cortical plate in P0 E24 MAM-treated animals). Since we know that migration of cortical neurons is impaired in MAM-treated animals, this analysis allowed us to compare the number of cells that migrated into the cortical plate close to and distant from the fluorescent microsphere injections in MAM-treated cultures. The values presented in our analysis represent the fraction of neurons that was able to migrate successfully into the first 60 μm zone (n = 4). The percent value was used in order to normalize across different cultures.
Effects of Conditioned Media on Disorganized Radial Glia
Injections of MAM into pregnant ferrets on E24 cause an assembly of effects, which include disorganization of radial glial cells. Culture of normal P0 cortical plate adjacent to E24 MAM-treated cortex improves the morphology of radial glia so that they realign in a more normal position (Hasling et al., 2003). The radial glia are identified in organotypic cultures or acute slices by small injections of fluorescent dextrans into the intermediate zone. We showed previously that such injections selectively label small populations of radial glial cells that colocalize with vimentin immunoreactivity (Juliano et al., 1996; Noctor et al., 1999).
To determine if the activity causing realignment of radial glia in E24 MAM-treated cortical plate could be isolated, conditioned media was obtained by growing normal P0 cortical organotypic cultures for 2 days and collecting the media. To apply a focused and directed source of the conditioned media, small aliquots were incubated with fluorescent microspheres and injected into the cortical plate of organotypic cultures of E24 MAM-treated brains. This method allows a slow release of the substance adhered to the microspheres (Riddle et al., 1997). We observed that fluorescent microspheres with adhered conditioned media were able to reorient radial glial cells toward their normal orientation when compared with injections in E24 MAM-treated slices with no additional treatment (Fig. 2). Fluorescent micro-spheres alone (control microspheres) with no additional media were not effective in reorienting radial glia toward their normal morphology. To quantify this finding we used the calculation described in Materials and Methods in which the angles of processes labeled after injections of Fluororuby were measured to give us a mean angular deviation that was not different from that observed in normal cultures. This measurement was significantly different from E24 MAM-treated slices cultured alone (Fig. 3).
Effects of Conditioned Media Fractionated into Different MWs on Disorganized Radial Glia
To further characterize the substance found in the normal cortex, we submitted conditioned media from normal slices to fractionation, using centrifugation. This allowed us to subdivide the conditioned media into aliquots of different MWs: <10 kDa, 10–30 kDa, 30–50 kDa, >50 kDa. Each MW fraction was incubated with fluorescent microspheres, which were then injected into organotypic cultures of E24 MAM-treated cortex. After 2 days of culture, each injected slice received one or more injections of Fluororuby. We found that the only MW fraction successful in causing the disrupted radial glia to realign toward their normal morphology was that of 30–50 kDa. This can be seen in Figures 3 and 4, which demonstrate the angular deviations of dextran injections related to different MW fractions and show images of the Fluororuby label in relation to the fluorescent microspheres. Normal organotypic cultures were also subjected to dextran injections after 2 days in culture for comparison of the angular deviations obtained from normal, untreated brains.
The Effects of Protein Denaturation on the Active Fraction
We conducted additional studies to further resolve if the substance that helped to radialize the disrupted radial glial cells in our model was a protein. If we could demonstrate that protein denaturation interfered with the ability of the MW fraction that produced the strongest radialization of the injected fibers (30–50 kDa, the ‘active’ fraction), then we would feel confident that the isolated substance was a protein. To do this, we first subjected the conditioned media of the active fraction to heat, which destroys protein activity. The conditioned media was then incubated with the fluorescent microspheres, which were injected into organotypic cultures of E24 MAM-treated cortex. After 2 days in culture, the slices with fluorescent microspheres and control slices (i.e. containing either microspheres alone with no adhered media, or microspheres with the active fraction and no heat, or with no microspheres) received injections of Fluororuby. Analysis of these cultures revealed that the microspheres containing the heated active fraction were no longer able to cause the labeled radial glia to return toward their normal morphology (Figs 3 and 4).
Next, we subjected the active fraction to treatment with Proteinase K, which also destroys protein activity. Again the fluorescent microspheres were incubated with the Proteinase K-treated active fraction and injected into the cortical plate of E24 MAM-treated and control slices. After 2 days in culture, and injection with fluorescent dextrans, we observed that the radial glia were not restored to their normal position (Figs 3 and 4). These experiments demonstrate that a protein-like molecule is involved in mediating the elongating effect on radial glia.
Effect of the Active Fraction on Neuronal Migration into the Cortical Plate
In organotypic cultures of E24 MAM-treated slices, or in E24 MAM-treated brains in vivo, cells do not migrate normally and remain in subcortical clusters, or in the intermediate zone (Noctor et al., 1999). For the next set of experiments, we observed whether the active fraction was able to improve migration of neurons into the cortical plate in conjunction with the realignment of radial glia. To do this, BrdU was injected into P0 ferret kits prior to preparation of the organotypic cultures. After the BrdU containing E24 MAM-treated cultures were prepared, injections of the active fraction adhered to fluorescent microspheres were made into the cortical plate. The cultures remained for 2 days, at which time the slices were reacted for BrdU immunoreactivity. The presence of the active MW fraction of conditioned media in the cortex improved migration near the treated microspheres (Figs 5 and 6). More cells generated on the day of culture preparation migrated into cortical plate in the vicinity of the microspheres than in more peripheral portions of the culture. In an individual cultured slice, therefore, the number of BrdU positive cells in the cortical plate diminished with distance from the microspheres (Fig. 6). The presence of the appropriate ‘factor’ improves migration into the neocortex.
In the model of cortical dysplasia achieved with MAM treatment on E24, several features of cortical organization are disrupted (Noctor et al., 1999). Radial glia are disorganized and demonstrate early astrocytic differentiation. In addition, Cajal Retzius cells are distributed throughout the cortical plate and intermediate zone, and the cortical plate is thin and poorly developed. These features of disruption (disorganized radial glia and Cajal Retzius cells) are key elements of normal cortical development and probably contribute to impaired neuronal migration in this model. The radial glia, long described as an essential scaffold for neurons migrating into the cortical plate (Rakic, 1990), are no longer functioning as effective guides. The disorganized Cajal Retzius cells, which produce Reelin, a protein that provides positional information to migrating cells, may provide improper cues to approaching cells (Rice and Curran, 2001). In an earlier study, we demonstrated that applying a focal source of factors present in normal newborn ferret cortical plate causes the radial glia to orient more appropriately (Hasling et al., 2003). In addition, this treatment causes Cajal Retzius cells to move into their normal position within the marginal zone and causes neurons to migrate more effectively into the cortical plate, rather than being stranded before reaching their target. We suggest that the most important element in the sequence of events that normalizes the MAM-treated cortical architecture is the improved phenotype of the radial glia. Providing a mechanism that encourages radial glial cells to elongate and assume a relatively normal position allows for reorganization of the Cajal Retzius cells and subsequent improved migration of cortical neurons.
The set of experiments reported here brings us closer to characterizing the nature of a ‘radializing factor’, which appears to be present in normal cortical plate. We demonstrate that a protein of 30–50 kDa nominal MW can be extracted from conditioned media of normal cortex; this protein has the capability to affect the morphology of radial glia. Hunter and Hatten (Hunter and Hatten, 1995) first proposed the existence of a factor capable of altering the morphology of radial glia. They observed that when astrocytes were exposed to a presumed soluble factor from immature neocortex, they resumed an elongated radial glial morphology. Exposure of astrocytes to older neocortex did not produce the same effect. Hunter-Schaedle (Hunter-Schaedle, 1997) subsequently reported that the molecule isolated to conduct the radializing activity was a protein of ∼55–60 kDa. Other studies support the notion that a factor with the ability to reversibly alter the morphology of radial glia or to elongate astrocytic processes is present in normal brain (Soriano et al., 1997; Leavitt et al., 1999; Leprince and Chanas-Sacre, 2001).
It is not yet known what the radializing substance is, whether there are multiple substances, or if we are characterizing the same substance identified by Hunter-Schaedle. We present evidence that this substance is a protein in a range of 30–50 kDa, which is similar to the size protein described by Hunter-Schaedle, and that it is a soluble molecule. Although this 30–50 kDa media fraction significantly improved the radial glial morphology in our study, the level of significance is not as great as that seen in the conditioned media alone. Since the action of the media fraction was not as effective as total conditioned media, this may imply that there is more than one substance or ‘factor’ capable of changing the morphology of radial glial cells.
Several specific substances appear to play a role in organizing the morphology of radial glia. Glial growth factor (GGF), a soluble form of neuregulin (NRG), is a molecule that occurs in a number of different isoforms, some of which fall in the size range of the protein reported here (Lemke, 1996). GGF/NRG plays a number of roles in development and has been reported by Anton and colleagues to be important in neuronal migration and for maintaining the shape of radial glial cells during cortical development (Anton et al., 1997; McGrath et al., 2001). GGF/NRG is known to act on the erbB family of receptors (Lemke, 1996; Anton et al., 1997). Since this molecule is present in immature neurons of the cortical plate, and various forms of the erbB receptors are present on radial glia and in the cortical plate, it is a likely candidate for a radialization molecule.
The biological activity important for communicating the radial glial phenotype, such as GGF/NRG, may arise from the migrating neurons themselves. Anton et al. (Anton et al., 1997) report that GGF is strongly expressed in migrating neurons, which may provide important information to maintain the radial glia phenotype. The migrating neurons in turn, may also receive feedback from the radial glia, which together provide a framework for maintaining both radial glial phenotype and the rate and direction of migration. In the MAM-treated animals, we may eliminate a population of neurons that provide morphologic cues to the radial glia while they move toward the cortical plate. By supplying the relevant molecule with the appropriate MW fraction of conditioned media, the appropriate morphology is restored. The exact distance that this diffusible molecule is active is not yet defined. Our observation that placement of the appropriate MW fraction in the cortical plate, or slightly deeper in the intermediate zone, are both effective in reorganizing the radial glia suggests that the molecule is active over a distance of at least a few hundred microns. Our earlier study, using explants of cortical plate co-cultured adjacent to MAM-treated slices, also implies that the active molecule is effective over some distance (Hasling et al., 2003). If injections of dextrans, which labeled the radial glia, were displaced from the microsphere injections, whether the labeled radial glial cells assumed an elongated morphology depended on the distance of the dextran injection from the microspheres. The finding that dextran injections some distance away from the protein source do not return radial glia toward normal morphology is supported by the observation in our earlier study that culturing a MAM-treated slice a few millimeters away from a normal slice is also not effective in restoring an elongated phenotype (Hasling et al., 2003). Taken together, these findings suggest there is a limit to the bioactive distance of the substance.
Several studies report that the protein Reelin may play a role in regulating radial glial phenotype. The name of this protein arises from the reeler mouse, which does not express Reelin due to a naturally occurring autosomal recessive mutation (D’Arcangelo et al., 1995). In addition to disorders in neuronal migration, reeler mice display abnormal radial glia with early differentiation into astrocytes (Caviness and Rakic, 1978; Hunter-Schaedle, 1997). The Reelin signal that normally delivers these cues is produced in the Cajal Retzius cells (D’Arcangelo et al., 1995; Ogawa et al., 1995). Because reeler mice lack the protein Reelin, several researchers considered that its absence might play a role in radial glial fate and a series of experiments support that idea (Soriano et al., 1997; Super et al., 2000). Other studies report that Reelin does not specifically alter the morphology of radial glial cells (Hunter-Schaedle, 1997). Although Reelin may not have a direct effect on radial glial cells, it might still play a role in causing radial glia to alter their morphology by downstream signaling or through its interaction with specific receptors. For example, Reelin has been proposed to act on integrin receptors (Dulabon et al., 2000). Integrin receptors appear to be important in mediating radial glial phenotype, since mice lacking the β1 integrin receptor also show early differentiation of radial glia into astrocytes (Dulabon et al., 2000; Magdaleno and Curran, 2001; McGrath et al., 2001).
Reelin is a much larger molecule than the putative factor identified here, but may play a role in radial glial morphology by its interaction with integrin (or other) receptors, which in turn regulate radial glial phenotype, perhaps through other downstream interactions. Support for this idea was recently published by Foerster et al. (Foerster et al., 2002), who observed that Reelin induced outgrowth of GFAP positive cells was lost in Dab1-deficient or β1 integrin-deficient mice. Integrin receptors may be a factor in maintaining the pial basement membrane (Magdaleno and Curran, 2001). This structure appears to be an important component of maintaining the integrity of the radial glial phenotype (Magdaleno and Curran, 2001; Halfter et al., 2002).
Our study supports the idea that one or more proteins are secreted by normal cortical plate, which maintains the radial glial phenotype during migration of neurons. We demonstrate that the addition of a 30–50 kDa protein obtained from normal cortex to E24 MAM-treated cultures improves the morphology of radial glia toward their normal appearance and increases the number of neurons that migrate into the cortical plate. We suggest that the realignment of radial glia allows the Cajal Retzius cells to reposition themselves (Hasling et al., 2003), which together provide a condition that enhances neuronal migration.
The work presented here was supported by NIH-RO1-MH-62 721 (S.L.J.). The authors acknowledge the technical assistance and excellent animal husbandry of Donna Tatham and also wish to thank Dr T.A. Hasling for assistance with several of the experiments.