Extensive migration of young neurons into the infant human frontal lobe

The first few months after birth, when a child begins to interact with the environment, are critical to human brain development. The human frontal lobe is important for social behavior and executive function; it has increased in size and complexity relative to other species, but the processes that have contributed to this expansion are unknown. Our studies of postmortem infant human brains revealed a collection of neurons that migrate and integrate widely into the frontal lobe during infancy. Chains of young neurons move tangentially close to the walls of the lateral ventricles and along blood vessels. These cells then individually disperse long distances to reach cortical tissue, where they differentiate and contribute to inhibitory circuits. Late-arriving interneurons could contribute to developmental plasticity, and the disruption of their postnatal migration or differentiation may underlie neurodevelopmental disorders.

to the portion of the cingulate (a subregion of Brodmann Area 24) contained in these tissue blocks as a "cingulate segment" (fig. S9A, blue shaded region). From each brain, one cingulate segment was cut on a cryostat into 30-micron sections that were used for quantifications. Since our specimens were cut into tissue blocks manually by hand, these tissue blocks were often variable in thickness and yielded different numbers of sections for analysis. To account for this variability and facilitate comparisons of cell numbers across specimens, we normalized our cell counts (obtained either through exhaustive counting or the Optical Fractionator, as described below) to a standardized segment arbitrarily defined as 150 sections or 4.5 mm. (equation 1).
(1) Normalized Cell Count = (!"## !"#$%)(!"#$%#&! !"#$%&' !"#$) !"#$%& !"#$%&' !"#$ The actual span for each cingulate segment was determined by the intersection interval multiplied by number of sections sampled. Sections were chosen for analysis using systematic random sampling. On a given coronal section, the cingulate gyrus was identified by its position on the medial aspect of the cerebral cortex, immediately above the corpus callosum and below the superior frontal gyrus. In Neurolucida or Stereo Investigator software, a contour was drawn around the entire pial surface of the cingulate. The lateral border of the contour was defined by the base of the cingulate sulcus extended perpendicularly to the corpus callosum. The boundary between the cingulate cortex and white matter was delineated using CTIP2 fluorescent staining in specimens younger than 5 months and NeuN fluorescent staining in specimens 5 months and older.
DCX, DAPI, and NeuN population sizes within a cingulate segment were estimated using the Optical Fractionator probe in Stereo Investigator. Unless stated otherwise, one brain was analyzed per age (N=1). Parameters for the Optical Fractionator study are given in Table S3. 3-micron guard zones were used, along with counting frame dimensions of 50x50 microns for DAPI and DCX and 75x75 microns for NeuN. 3 to 6 sections were analyzed per brain. Range of sampling sites per brain: DAPI (cortex), 70-260; DAPI (white matter), 50-105; NeuN, 130-330; DCX: 325-930. Individual cells were counted using standard stereologic methods with a 63x oil immersion lens on a Zeiss Axioscope II epifluorescent microscope. Adequate sampling was monitored by calculating coefficients of error (Gundersen m=1), with all C.E.'s <0.1.
Interneuron subtype cell numbers in a cingulate segment were estimated using an exhaustive counting approach, as their scarcity at certain ages and in some regions (e.g. white matter) made an Optical Fractionator study impractical. Cells were counted exhaustively on tiled images of fluorescently labeled sections, acquired at 10x magnification. Neurolucida was used to keep track of both the total number of cells counted and their spatial distribution. Total interneuron cell number per cingulate segment was then calculated by summing the exhaustive cell counts across all sections and then correcting for the section sampling fraction and normalizing to a standardized segment of 150 sections, as described above. 3 to 6 sections were used per brain.
Section sampling frequencies for subtype counts are given in Table 4.
For counts on the subpallial transcription factors near the ventricular wall and within the Arc, 3 equivalent fields of view were imaged for each region, using a 40x lens on a Leica white light confocal microscope, and quantified. 3-4 individual neonatal cases were used for a total of 9-12 samples per count.
Calculation of cingulate volumes. The Cavalieri Estimator in Stereo Investigator was used to calculate the volume of cingulate segments at each age. Cingulate segments were collected as described above for cell quantifications and as depicted in fig. S9A. Ten sections, spaced 15 sections apart were chosen for analysis. Mounted section thickness was 30 microns. Grid size was 500 microns. Coefficients of error (Gundersen m=1) were <0.04 for all specimens analyzed.
Organotypic slice cultures and live imaging. When autopsy was performed with a postmortem interval less than 18hrs, tissue was collected for live-imaging experiments. The fresh, unfixed, tissue was cut either in a coronal or sagittal block that was less than 1cm thick. It was kept in cold artificial cerebrospinal fluid (ACSF) that was oxygenated until embedded in 3.5% low-melting-point agarose (Fisher) and sectioned using a Leica VT1200S vibrating blade microtome to 250-300µm slices in ACSF containing 125 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 and 1.25 mM NaH 2 PO 4 . The sections were then transferred to Millicell-CM slice culture inserts (Millipore) that were immersed in cortical slice culture medium [66% BME, 25% Hanks, 5% FBS, 1% N-2, 1% penicillin, streptomycin, and glutamine (all Invitrogen) and 0.66% D-(+)-glucose (Sigma-Aldrich)]. An adenovirus (AV-CMV-GFP, 1 × 10 10 ; Vector Biolabs) at a dilution of 1:50-1:500 was applied to the slices, which were then cultured at 37°C, 5% CO 2 , 8% O 2 . For time-lapse imaging, cultures were then transferred to an inverted Leica TCS SP5 confocal microscope with an on-stage incubator streaming 5% CO 2 , 5% O 2 , and balanced N 2 into the chamber. Slices were imaged using a 10× air objective at 25 min intervals for up to 3 d with repositioning of the z-stacks every 6-8h. For post hoc analysis, slices were fixed in 4% PFA and processed as floating sections for immunohistochemistry as described above.
MRI. The images were acquired on a full body GE MR950 7T scanner using a 32-ch Nova Medical head coil with 3D fast spin echo sequence, isotropic 600-micron resolution, time to echo (TE) of ~120ms and TR of 2.5s, and 8 averages. The scan time was approximately 30 minutes.
The distribution of migratory cells were manually labeled using MNI-Display software (http://www.bic.mni.mcgill.ca/ServicesSoftwareVisualization/Display). The cells on the MRI images appear distinctively darker than white matter intensity, and look similar to the intensity of gray matter. Previous studies had recognized a periventricular signal that was distinct from that of the overlying developing white matter (44,45). The rater works mainly on the sagittal plane, and trimmed or added segmentation using the axial and coronal views if necessary. One rater (M.P.) segmented all cases and reviewed the resulting labels with H.K. The rater repeated segmentation with a one-month time distance. The intra-rater reproducibility was excellent as the Quantitative analyses on MRI. We measured two different characteristics of the migratory cell distribution: extent and location of the cell distribution. To measure the extent, we first skeletonized the whole label using a mathematical solution developed previously (46). This algorithm further created a distance map from one end to the other along the skeleton. The maximum distance between the two ends of the skeleton was normalized by dividing it with the whole extent of the ventricle, and was used for the measurement of extent. Regarding the location of the cell distribution, we were interested in how the distribution was located with respect to the lateral ventricle in the anterior-posterior direction. To assess this for each individual, we computed the centroids of the label volume and of the lateral ventricle. We computed the distance between these two centroids in y-axis (i.e., anterior-posterior direction), and normalized it using the distance between the anterior and posterior tips of the ventricle. We used this normalized value to assess the location of cell distribution.                Movie S1: 3D rendering of DCX+ cells (green) in Arc strip ensheathed by GFAP+ fibers (pink) at 1.5 months.
Movie S2: Time-lapse imaging showing a migrating neuron in a sagittal cortical slice from a brain at birth. Area imaged is developing white matter of cingulate. Note cell (*) is traveling in anterio-dorsal direction. Movie spans 18 hours.
Movie S3: Time-lapse imaging showing a migrating neuron in a coronal cortical slice from a brain at birth. Area imaged is at the dorsolateral edge of the lateral ventricle, within the Arc region. Movie shows a migrating neuron (*) leaving the dense cell collection. Movie spans 24 hours.
Movie S4: Time-lapse imaging showing migrating neuron in a coronal cortical slice from a brain at birth. Area imaged is at the dorsolateral edge of the lateral ventricle, within the Arc region. Movie shows migrating neuron (*) traveling alongside dense cell collection. Movie spans 24 hours.
Movie S5: Schematic animation of three-dimensional rendering of postnatally migrating neurons in the infant brain. Arc-orange, SVZ and RMS-magenta, MMS-green.