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Interneurons in the Developing Human Neocortex

Nada Zecevic, Frances Hu, Igor Jakovcevski

Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut 06030

Received 16 April 2010; accepted 2 June 2010

ABSTRACT: Cortical interneurons play a crucial interneuron progenitors that have neocortical origin.
role in the functioning of cortical microcircuitry as they Neuropeptide Y, somatostatin, or parvalbumin cells are
provide inhibitory input to projection (pyramidal) neu- sparse in mid-term cerebral cortex. In addition to the
rons. Despite their involvement in various neurological early source of cortical interneurons in the GE and later
and psychiatric disorders, our knowledge about their in the neocortical SVZ, other regions, such as the subpial
development in human cerebral cortex is still incomplete. granular layer, may also contribute to the population of
Here we demonstrate that at the beginning of corticogen- human cortical interneurons. In conclusion, our findings
esis, at embryonic 5 gestation weeks (gw, Carnegie stage from cryosections and previous in vitro results suggest
16) in human, early neurons could be labeled with calreti- that cortical interneuron progenitor population is more
nin, calbindin, and GABA antibodies. These immunola- complex in humans relative to rodents. The increased
beled cells show a gradient from the ganglionic eminences complexity of progenitors is probably evolutionary adap-
(GE) toward the neocortex, suggesting that GE is a well tation necessary for development of the higher brain
conserved source of early born cortical interneurons functions characteristic to humans. ' 2010 Wiley Periodicals,
from rodents to human. At mid-term (20 gw), however, a Inc. Develop Neurobiol 71: 18–33, 2011
subset of calretinin+ cells proliferates in the cortical Keywords: cortical interneurons; human fetal brain;
subventricular zone (SVZ), suggesting a second set of immunohistochemistry; cerebral cortex

INTRODUCTION trophysiological properties (e.g., Markram et al.,

2004; Wonders and Anderson, 2006, Butt et al.,
Two types of neurons: projection and interneurons 2008). On the basis of their calcium-binding proteins
are both necessary to establish complex neural and neuropeptide expression, interneurons can be
network and proper function of the cerebral cortex. subdivided into partially overlapping categories of
Projection neurons are pyramidal in shape, contain cells that express calbindin (CB), calretinin (CalR),
glutamate, and are excitatory in action, whereas inter- parvalbumin (PV), vasoactive intestinal peptide,
neurons are small local circuit neurons, mostly neuropeptide Y (NPY), cholecystokinin, somatostatin
GABAergic and inhibitory. Cortical GABAergic (Sst), and choline acetyltransferase (e.g., Markram et
interneurons are highly diverse and classified accord- al., 2004; Xu et al., 2004; Wonders and Anderson,
ing to a variety of morphological, antigenic, and elec- 2006; Butt et al. 2008; Gonchar et al., 2008). Under-
standing how interneuron diversity emerges during
Correspondence to: N. Zecevic (
development is crucial for a better understanding of
Contract grant sponsor: NIH NS 041489-09; contract grant num- normal cortical function as well as numerous neuro-
ber: 41489-09. developmental disorders. Cortical interneurons have
' 2010 Wiley Periodicals, Inc. a role in normal development through their influence
Published online 30 November 2010 in Wiley Online Library
on cell proliferation and migration (Haydar et al.,
DOI 10.1002/dneu.20812 2000; Owens and Kriegstein, 2002). Importantly,
Human Fetal Cortical Interneurons 19

cortical interneurons are implicated in neuropsychiat- (Ulfig, 2002), not present in rodents. This is why
ric disorders that range from cortical ectopias with throughout this article we use terms GE and neocor-
epilepsy (DeFelipe, 1999; Gleeson and Walsh, 2000) tical VZ/SVZ, instead of ventral telencephalon
to schizophrenia, autism, and bipolar disorder (pallidum) and dorsal telencephalon (pallium), which
(Akbarian et al., 1995; Knable, 1999; Lewis and Lev- are commonly used in reports on rodents.
itt, 2002; Levitt, 2003; Baraban and Tallent, 2004; Another specificity of the human fetal brain is the
Lewis et al., 2005). transient cellular layer, the subpial granular layer
In rodents most, if not all of the cortical interneur- (SGL), below the pia, which may generate an inward
ons, originate in the ganglionic eminence (GE) of the migrating subtype of cortical interneurons in primates
ventral telencephalon, and migrate tangentially to the (Zecevic and Rakic, 2001; Rakic and Zecevic, 2003).
dorsally located cerebral cortex (Anderson et al., Our findings in the human fetal brain argue for a
1997, 2001; Tamamaki et al., 1997; Parnavelas et al., more complex interneuronal progenitor population
2000; Marin and Rubenstein, 2001). Different sub- and various sites of origin of cortical interneurons
populations of interneurons have distinct ventral relative to rodents. Although rodent brain is an excel-
origins: PV-expressing (PV+) and Sst+ cells originate lent model and continues to provide important data
from the Nkx2.1 lineage in the medial GE (MGE), with recent advances in genetics and molecular biol-
CalR+ cells originate mainly in the caudal GE (CGE), ogy, it needs to be complemented with studies of the
whereas CB+ cells originate throughout the GE (Xu human brain. More knowledge about human cortical
et al., 2004; Gonchar et al., 2008). interneurons is necessary for understanding how
In contrast to rodents, the long developmental the initial cortical circuitry is established as well as to
period and a larger human brain with new cortical devise future therapeutic approaches to disorders
areas and expanded upper cortical layers (Hill and characterized by malfunction of GABAergic signal-
Walsh, 2005; Molnár et al., 2006; Rakic, 2009) is ing (e.g., epilepsy, psychiatric disorders).
likely to need additional sources of interneurons in
late corticogenesis. The neocortical subventricular
zone (SVZ) evolutionary expands in primates (Smart METHODS
et al., 2002; Lukaszewicz et al., 2006) and even more
in the human brain (Zecevic et al., 2005, Bayatti Human Fetal CNS Tissue
et al., 2008). This additional proliferative zone is a
source of late born cortical neurons, including inter- Human fetal brain tissue was obtained from the Human
Fetal Tissue Repository at the Albert Einstein College of
neurons. Although not conclusive, several lines of
Medicine (Bronx, NY), after legal abortions, with proper
evidence support the dual origin of cortical inter-
consent from parents. Handling of human material was
neuron in primates (Letinic et al., 2002; Rakic and done with special care following all necessary requirements
Zecevic, 2003; Fertuzinhos et al., 2009; Petanjek and regulations set by the Ethics Committee of the Univer-
et al., 2009). The idea that primates have a more sity of Connecticut and the Helsinki Convention. In all
diverse interneuronal population relative to other studied cases, ultrasound and gross neuropathological
mammals including rodents, came from reports on examination confirmed that the brain tissue was normal.
various interneuronal cell types, such as well devel- Brain tissue was placed in medium containing modified
oped double-bouquet cells, their possible ventral and Hank’s Balanced Salt Solution (Sigma, St. Louis, MO),
dorsal origin and molecular characteristics in the 2 mM glutamine (Invitrogen, Carlsbad, CA), 10 mM
human brain (Gabbott et al., 1997; DeFelipe, 2002; HEPES buffer (Sigma), and 20 lg/mL gentamycine sulfate
and transported to our institution, where it was immediately
Letinic et al., 2002; Preuss and Coleman, 2002; Rakic
dissected into coronal blocks for freezing and subsequent
and Zecevic, 2003; Yanez et al., 2005; DeFelipe
immunohistochemistry, or for cell cultures. Blocks of tissue
et al., 2006; Jones, 2009; Zaitsev et al., 2009). In this were fixed in 4% paraformaldehyde in 0.1 M phosphate
study, we provide additional results that support dual buffer (pH 7.4) overnight, cryoprotected in 30% sucrose,
origin of cortical interneurons. The terms \ventral" frozen in precooled 2-metylbutane, and stored at 708C
and \dorsal" for GE and cortex, respectively, are until sectioned (15-lm thick) in the frontal or sagittal plane
commonly used referring to rodent brains in which and processed for immunohistochemistry.
GE is indeed positioned ventrally to the cerebral cor-
tex. In the large human brain, the newly developed
temporal lobe, with amygdala, hippocampus and the Immunohistochemistry
abutting neocortical SVZ, is however, also ventrally Various primary and secondary antibodies (Table 1) were
positioned. This part of the brain may be supplied used at optimal dilutions. Cryosections were incubated in
with interneurons from the inferior part of the GE blocking solution [1% BSA (Sigma), 5% normal goat serum
Developmental Neurobiology
20 Zecevic et al.

Table 1 Primary Antibodies Used in This Study

Antigen Host Clone Dilution Manufacturer Catalog No.
Somatostatin Rat IgG 1:100 Millipore Chemicon MAB354
Parvalbumin Mouse IgG1 PARV-19 1:100 Sigma P3088
Calretinin Mouse IgG1 1:100 Chemicon MAB1568
Calretinin Rabbit IgG 1:2000 Swant, Belliziona, Switzerland 7696
Calbindin Mouse IgG CB-955 1:2000 Sigma C9848
Calbindin Rabbit IgG 1:5000 Sigma C7254
GABA Mouse IgG GB-69 1:1000 Sigma A 0310
GAD65 Mouse IgG 1:100 Millipore Chemicon MAB351
GAD67 Rabbit IgG 1:1000 Chemicon AB108
Nkx2.1 (TTF1) Rabbit IgG EP1584Y 1:1000 Epitomics 2044–1
panDlx (Dll) Rabbit IgG 1:1000 Gift from Dr. Y. Morozov, Yale Univ.,
New Haven, CT.
NPY Rabbit IgG 1:5000 ImmunoStar, Hudson, WI 22940
Mash1 Mouse IgG 1:500 BD Pharmingen 556604
Phospho-histone-H3 Rabbit IgG 1:500 Upstate Biotechnology 06–570
Ki67 Mouse IgG 1:500 Gift from Dr. Noctor, Columbia University,
Ki67 Rabbit IgG 171018 1:500 Abcam ab833

(Vector Laboratories, Burlingame, CA), and 0.5% Tween observed and delineated under low-power magnification
20 in phosphate buffered saline] for 30 min at room temper- (103 objective) with a 365/420 nm excitation/emission fil-
ature. Primary antibodies were applied overnight at 48C, ter set (Zeiss, blue fluorescence). The nuclear staining
whereas corresponding fluorescein- or rhodamine-tagged allowed delineation of areas of interest (e.g., GE, cortical
secondary antibodies were subsequently applied for 2 h at plate, etc.). For various transcription factors, calretinin and
room temperature. For double and triple staining, the Ki67, two-dimensional counts of the labeled cells/nuclear
primary antibodies were mixed at optimal dilutions and profiles were performed under 403 objective in each
subsequently detected using mixtures of appropriate delineated field and profile number was normalized to area.
secondary antibodies (multiple-labeling grade antibodies, In addition, the number of immunolabeled cell profiles
Jackson ImmunoResearch, West Grove, PA). Nuclei were normalized to overall cell number (number of nuclear pro-
counterstained with short (5 min) incubation in 1% bis- files) was calculated, and will be further referred to as a
benzamide (Polysciences, Warrington, PA). For enzyme- \relative density". In large midgestational brains, a grid
substrate immunohistochemistry the procedure was essen- (60 3 60 lm) was placed over area of interest and random
tially identical, except that at the beginning the endogenous samples 120-lm apart were counted and averaged for
tissue peroxidase activity was blocked for 20 min by 0.3% overall area. At least three sections per case were counted.
H2O2 solution in methanol, and the incubation with biotin- We would like to stress that in some cases when we
conjugated secondary antibody was for only 30 min at doubted the specificity of immunostaining, or when parts of
room temperature. Immunoreactivity was revealed with the sections were missing and/or appeared damaged during
Vectastain ABC Kit (Vector Laboratories) by using 0.05% tissue processing we did not include results in our analysis.
diaminobenzidine as a substrate, according to manufac- Because of a very limited sample size we did not consider
turer’s instructions. For each antigen, specificity of staining applying stereological methods, and we offer our quanti-
was tested by omitting the first antibody, or replacing it fications just as an estimate and look forward to further
with corresponding normal serum at the same concen- studies on more cases to confirm our results.
tration. In addition, analysis was performed only on those
sections in which the morphology of the labeled cells
appeared to be characteristic of the cell type expected to be
labeled, and the staining pattern was consistent with the
Image Analysis and Photographic
subcellular distribution of the antigen (e.g., transcription Documentation
factors within nucleus, receptors on the membrane). Photographic documentation was done using a confocal
laser-scanning microscope (Carl Zeiss, LSM 510), Axio-
Cell Quantification plan fluorescent microscope (Carl Zeiss), and Axioskop
brightfield microscope (Carl Zeiss). Image processing was
The counts were performed on an Axioskop microscope done by Adobe Photoshop CS2 software (Adobe Systems,
(Zeiss, Oberkochen, Germany) equipped with a motorized San Jose, CA). Images were modified with regard to bright-
stage and Neurolucida software-controlled computer sys- ness/contrast in order to get the nearest representation of
tem (MicroBrightField, Colchester, VT). Sections were the image seen through the microscope oculars.
Developmental Neurobiology
Human Fetal Cortical Interneurons 21

Figure 1 Expression of calretinin (CalR) and calbindin (CB) in the 6- to 7-week-old embryo.
Whole parasagittal sections through embryonic forebrain immunostained for CalR (A–C) and CB
(D–F). B, C and E, F represent enlarged images from squares in A and D, respectively. VZ, ventric-
ular zone; PPL, primordial plexiform layer; GE, ganglionic eminence; o.b., olfactory bulb; hipp,
hippocampus. Scale bars: 2 mm (A, D), 25 lm (B, C, E, F).

RESULTS stage, CS 16) are reported in the primordial plexiform

layer (PPL), an early structure that is divided by the
Interneuronal Markers in the Human emerging cortical plate around the 8th gw into layer I
Embryonic Brain (5–8 gw) (upper part) and the subplate layer (SP, lower part).
At 6 gw (CS 17) neurons in the PPL and the GE
Several established interneuronal markers are express two calcium-binding proteins, CalR and CB,
expressed in the human cerebral cortex from early but not PV or Sst (Figs. 1 and 2). The density of
embryonic stages (see Zecevic and Milosevic, 1997; immunolabeled cells with either of these two markers
Zecevic et al., 1999; Rakic and Zecevic, 2003). Cells is the greatest in the matrix of the GE, which at this
expressing GABA, CalR, and CB at 5 gw (Carnegie stage is not yet divided into LGE and MGE. There is
Developmental Neurobiology
22 Zecevic et al.

Figure 2 Calretinin (CalR) and calbindin (CB) cells in the human embryonic forebrain are
migrating young neurons. (A) A mid-sagittal section through the brain of 6 gw embryo. (B) CalR
and (C) CB label cells in the mantle zone of ganglionic eminence (GE). (D) Higher magnification
of the ventral telencephalic area boxed in B, CalR+ cells make a \touchdown" before continuing to
dorsal neocortical primordial plexiform layer (PPL). (E) CalR and (F) CB cells in the PPL. LV, lat-
eral ventricle.

a clear ventro-dorsal gradient of immunoreactivity [Fig. 2(D)]. At a similar embryonic age (7 gw, CS
for both CalR and CB from the GE toward the emerg- 19), among tangentially migrating cells, occasional
ing neocortex (Figs. 1 and 2). Importantly, all radially oriented cells within the telencephalic wall
immunolabeled cells were demonstrated outside of could be double-labeled with neuronal markers (b-III
the proliferative ventricular zone, consistent with tubulin or GABA) and ventral transcription factors,
labeling of young interneurons, and not their pro- Nkx2.1 or Dlx2 (Rakic and Zecevic, 2003, Fig. 3).
genitors. Immunoreactive cells had the appearance The early presence of these double-labeled cells
suggestive of tangentional migration to the neocortex, extending from the VZ to the pia, is suggestive of
based on the orientation of their processes and their local, cortical origin.
their ventro-dorsal gradient (see Fig. 2). Notably, a At 8 gw (CS 20), the cortical plate (CP), emerges
stream of migrating cells appeared to come into close as initially only 1–2 cells rows thick layer dividing
contact with ventral brain surface, before \bouncing the PPL into upper and lower parts. From the very
off" to more dorsally located cortical primordium beginning, GABA+ or CalR+ neurons are present in
Developmental Neurobiology
Human Fetal Cortical Interneurons 23

Cortical Interneurons at 15–20 gw

During the next 2 months, from 8 to 15 gw, the num-
ber of CalR+ cells increases throughout the develop-
ing cerebral cortex. In sagittally cut sections through
the 15 gw brain, the density of CalR+ cells is higher
in occipital than in frontal cortical regions (see
Fig. 5), demonstrating a caudo-rostral gradient of
CalR expression. A similar caudo-rostral developmen-
tal gradient has been reported for calcium-binding pro-
tein expression in embryonic (Zecevic et al., 1999) and
fetal human cortex (Bayatti et al., 2008), as well as for
general cellular and functional maturation of the CNS
(e.g., Del Rio et al., 1995; Yan et al., 1997).
At 15 gw, relative density of CalR+ cells is still the
highest in the GE with much lower density calculated
Figure 3 Expression of Dlx2 and Nkx2.1 in embryonic in the cortical VZ/SVZ [Fig. 6(A)]. This distribution
cerebral cortex. (A) Drawing of horizontal section of em- suggests that at 15 gw, the GE is still the main source
bryonic 6–7 gw brain. (B) Nkx2.1 (red) is expressed in the of cortical CalR+ cells. At 15 gw, GE is divided into
nucleus of GABAergic cells (green) in the preplate (PPL).
LGE and MGE, with CGE at more caudal telence-
(C) Dlx2 (green) in the nucleus of a radially oriented neu-
ron (b-IIItub+) in the ventricular zone. Modified from
phalic levels. From the immunolabeling studies it is
Rakic and Zecevic, 2003, with permission. not clear where exactly in the GE do CalR+ or CB+
cells originate, since they are seen as postmitotic,
probably migrating cells.
the newly formed CP [Fig. 4(A)]. These early immu- In the neocortical VZ/SVZ, the percentage of
nolabeled cells will eventually become deep layers CalR+ cells from all cells increases approximately six
V-VI interneurons, as the inside-out formation of the times from only 1% at 15 gw to 5.9% at 20 gw
CP proceeds. On coronal sections from an 8-week- [Fig. 6(A)]. In layer I, both small cells and much
old embryo the width of the CP is seven times larger larger Cajal-Retzius cells are strongly reactive for
in the lateral (140 lm) than in the medial (20 lm) CalR at this stage [Figs. 6(E) and 7(D)], similar to
regions of the telencephalic wall. CalR+ cells are earlier fetal stages (Figs. 1 and 2). Relative density of
observed to follow the inside-out developmental gra- CalR+ cells in different forebrain regions also
dient, as described for projection neurons (see Fig. 4). changes and for the first time at mid-term becomes

Figure 4 CalR + cells in the emerging cortical plate at 8 gw. (A) In the medial cortex cortical
plate (CP) is only 2–3 cell rows wide (vertical bar 20.8 lm), and increases in width in (B) the dor-
sal (vertical bar 37.5 lm), and (C) lateral cortex (vertical bar 141.6 lm). Bisbenzamide stained cell
nuclei (blue). PPL, primordial plexiform layer; LyI, layer I; SP, the subplate layer; VZ, ventricular

Developmental Neurobiology
24 Zecevic et al.

not label CalR+ Cajal-Retzius cells, but in layer I,

30% of small CB neurons coexpressed CalR+; single
labeled cells for CalR and CB were also present
[Fig. 6(B,F)]. It is interesting to note that in all corti-
cal regions studied at mid-term, layer I was filled
with CalR+ puncta, reminiscent of axonal terminals
and/or dendrites [Fig. 5(F)]. In contrast to numerous
CalR+ cells in the cortical VZ/SVZ, only rare CB+ cells
could be detected in that region [Figs. 5(G) and 8], in
contrast to the GE, where both single CalR+, CB+, and
double-labeled cells were present (not shown).
Numerous CalR+ and CB+ cells were present in a
well developed GE positioned caudally and inferi-
orly, around the temporal extension of the lateral
ventricle [Fig. 9(C)]. A number of Ki67+ cells in the
inferior GE indicates cell proliferation in this region
at mid-term. This part of GE would correspond by its
position to the caudal GE (CGE) described in mice
(Nery et al., 2002; Kanatani et al., 2008).

Morphology of Cells Labeled with

Interneuronal Markers
The morphology of CalR+ and CB+ cells in the first
half of gestation in all cortical layers is mainly
bipolar, with one process on each cell poles. In the in-
termediate zone (IZ), the subplate (SP) and CP their
orientation is mostly radial, consistent with their
migration toward upper cortical layers (Figs. 6
and 7). Occasional transversally crossing cells can be
Figure 5 Caudo-rostral gradient of cortical CalR+ cells. seen at all cortical levels, similar to what was
(A). Drawing of a sagittal section through 15 gw fetal fore- described in our previous report (Zecevic et al.,
brain with boxed areas presented on pictures. (B) CalR 1999). The tangentally oriented CalR+ cells at mid-
immunoreactions (red) in the GE; (C, E) in the frontal cor-
term are demonstrated in the SVZ [Fig. 7(C)], and
tex and (D, F) in the occipital cortex. Blue- bisbensamide
occasionally even in the CP [Fig. 6(H), arrow]. The
staining of cell nuclei. Lay I, layer I; CP, the cortical plate;
VZ, ventricular zone; Cer, cerebellum; GE, ganglionic emi- majority of immunolabeled cells in the CP are, how-
nence. Scale bars: A, E, F-50 lm; C, D-50 lm. ever, radially oriented with a leading process oriented
either toward pia, or less often toward the SVZ
[Fig. 6(I), arrows]. Immunolabeled cells with ran-
higher in the cortical VZ/SVZ relative to the GE domly oriented processes or with two processes
[Fig. 6(A)]. In respect to the ratio of two Ca-binding branching from the upper side of the cell could be
proteins, CalR+ cells at mid-term outnumber CB+ seen in the CP [Fig. 7(C), arrow]. A number of CalR+
cells in all cortical regions except in deep layers V- cells could also be seen in close proximity to the
VI [Fig. 6(B,C)]. Some of the deep CB+ cells may ventricular surface [Figs. 7(D) and 10(A)].
represent a subpopulation of pyramidal cells. This ob- At mid-term, the three interneuronal markers,
servation is corroborated by the strong labeling of CalR, CB, and GABA were strongly expressed in the
axonal tracts, including the internal capsule that con- CP neurons, mostly in deeper layers, as well as in the
tains corticospinal axons [Fig. 6(D)]. Additionally, in proliferative SVZ [Fig. 8(A–D)]. Other interneuronal
the upper cortical layers (layers II-III) single CB+ markers such as PV, Sst, and NPY were expressed by
cells with large round cell bodies were labeled, as sparse cells in the subplate or intermediate zone, and
well as numerous radially spreading processes more often in the hippocampal region than in the
[Fig. 6(E)]. CalR+ cells are closely apposed to these neocortical areas [Fig. 8(E–G)]. Few NPY+ cells
CB+ processes, as if migrating along them. CB did were present also in layer I (not shown).
Developmental Neurobiology
Human Fetal Cortical Interneurons 25

Figure 6 Expression of CalR and CB in the human forebrain at 7–20 gw. (A) Relative densities
of CalR+ cells in the human ganglionic eminence (GE), cortical ventricular/subvetricular zones
(VZ/SVZ) and primordial plexiform layer (PPL) at 7 weeks or cortical plate at 15 and 20 weeks.
(B) Relative densities of CalR+, CB+, and CalR+/CB+ cells in layers I (lay 1), II-III (lay 2–3), and
layer V (lay 5) of a 20-week-old cortical plate. (C) CB+ cells (red) in the layer V of a 22-weeks-old
motor cortex. (D) CB+ axons (red) in the internal capsule. (E) In layer II of the lateral cortex
CalR+ (green) cells are apposed to the CB+ axons (red, arrows). Arrowheads point at CB+ cell
bodies. (F) Layer I of the lateral cortex double-stained for CalR (green) and CB (red). CalR+ Cajal-
Retzius cells (large arrow), a much smaller double-labeled CB+/CalR+ cell (yellow in overlay,
small arrow), CB+ cells (empty arrowheads), and a small CalR+ cell (filled arrowhead). (G) CalR+
(green) and CB+ (red) cells in the dorsal cortical ventricular zone. (H, I) In layer III of the cingulate
cortex at 20 gw. CalR+ cell extending leading process horizontally (H, arrow), or toward the ventri-
cle (I, arrows). Blue– cell nuclei counterstained with bisbenzamide in C, D, H, I. LV, lateral ventri-
cle. Scale bars: 25 lm (E–G), 20 lm (C, H-I), 50 lm (D).

Developmental Neurobiology
26 Zecevic et al.

Figure 7 CalR+ cells at various developmental ages. (A) At 7 gw in the PPL, (B) 15 gw, (C) 17
gw-CalR+ cells are mainly radially oriented toward pia. Occasionally they have two oblique proc-
esses or look as tangentially migrating cells (arrows); (D) Number of CalR+ cells increases at 20
gw in the SVZ and the layer I.

Progenitors of Cortical Interneurons in cortical SVZ. Notably, among various cell types that
the Neocortical SVZ at Mid-Term proliferate in the SVZ at mid-term (Zecevic et al.,
2005), is a subpopulation of CalR+ cells, as demon-
Neocortical SVZ at mid-term is still a very active
strated by their colabeling with a proliferation marker
proliferative zone that extends 800–1200 lm from
the ventricular surface, and can be divided into inner Ki67 (Fig. 10, Yu et al., 2009). We estimated that at
and outer SVZ [Fig. 9(A)]. Labeling with prolifera- 20 gw 17% of CalR+ cells in the cortical VZ/SVZ
tion marker Ki67 demonstrated that more cells are were proliferating, as they were colabeled with Ki67.
proliferating in the SVZ than in the VZ/SVZ of the On the other hand, CalR+ cells represent 10% of
GE, where cell proliferation is almost exhausted the total proliferating population in the SVZ at that
[Fig. 9(B)], suggesting that the main proliferative time. This finding obtained on cryosections comple-
zone for late born cortical neurons is the outer neo- ments earlier reports in vitro (Zecevic et al., 2005).
Developmental Neurobiology
Human Fetal Cortical Interneurons 27

Figure 8 Interneuron markers expressed at mid-term. (A) CB+ cells are grouped in deeper layers of
the CP, (B) the same section with bisbensamide staining of nuclei (blue); (C) GABA+ cells in the CP,
(D) Higher magnification of GABA+ cells in the CP; (E) multipolar Sst+ cells in the hippocampal
region; (F) sparse PV+ cells in the intermediate zone (IZ), (G) NPY+ cells in the subplate (SP) layer.

Combined, these results are consistent with the local 1999; Rakic and Zecevic, 2003). At 13 gw, a gradient
origin of a subpopulation of late born cortical CalR+ of CalR+ cells extends from temporal lobe, where it
cells in the human brain. consists of several rows of immunolabeled neurons,
to dorsolateral Cx, where it is only one cell thick
(Zecevic and Milosevic, 1997). At mid-term numer-
Subpial Granular Layer
ous CalR+ cells, Dll+, and a much smaller population
Cells of the transitory SGL in primates spread over of Nkx2.1+ and CB+ cells can be seen in the SGL
the entire neocortex (Brun, 1965; Meyer and Wahle, (Zecevic et al., 1999; Zecevic and Rakic, 2001; Rakic
Developmental Neurobiology
28 Zecevic et al.

finding shown in this study is the presence of prolifer-

ating CalR+ cells at mid-term as demonstrated on
cryosections by their double-labeling with the proli-
feration marker, Ki67. These double-labeled cells
represent, in our opinion, a novel type of cortical
progenitor cells previously unreported in either
rodents or primates (see below).

The Initial Appearance of CalR+

and CB+ Cells
In the human fetal brain CalR+ and CB+ cells are
present continuously from early embryonic stages,
before the appearance of the CP, to mid-term. On the
basis of their gradient in distribution, CalR+ and CB+
cells seem to be generated first in the MGE. In later
fetal stages, however, CalR+ cells are also generated
in the CGE, the inferior GE, and cortical SVZ. From
the GE first CalR+ and CB+ neurons in the embryonic

Figure 9 Proliferative zones at mid-term. (A) Large neo-

cortical VZ/SVZ (vertical bar 800 um) with dense Ki67+
proliferating cells (red) in the outer SVZ (oSVZ); (B) In the
ganglionic eminence (GE) fewer cells are labeled with
Ki67 (green) and are positioned close to the ventricle. (C)
Colabeling of CalR (red) and Ki67 (green) in the inferior
GE—boxed area in the drawing through caudal part of the
telencephalon. Th, thalamus; ic, internal capsule; LV, lat-
eral ventricle; Hipp, hippocampus.

and Zecevic, 2003). Numerous Dll+ small neurons

are GABAergic (Rakic and Zecevic, 2003), whereas
a different population of much larger cells is reelin+
Cajal-Retzius neurons [Fig. 6(F)].


In this study we summarize our results on antigen

characteristics, morphology, and distribution of
several classes of cortical interneurons and their pro-
genitors in human fetal cerebral cortex during the first
Figure 10 Cortical interneurons proliferate in the neo-
half of gestation. Using immunolabeling with a
cortical SVZ at mid-term. (A) CalR+ cells (red) in the VZ/
battery of interneuronal markers, we extended our
SVZ region occasionally touch the ventricular surface; (B)
previous results on the initial appearance of the A subpopulation of cells are double-labeled with CalR
GABA and calcium-binding proteins in the human (green) and proliferation marker Ki67 (red) in the cortical
cortical primordium (Zecevic and Milosevic, 1997; SVZ. (C) Optical sectioning confirmed that double-labeled
Rakic and Zecevic, 2003) with the results in fetal CalR+/Ki67+ cells are present in the mid-term SVZ. bb,
brains up to mid-term (20 gw). An important new blue, nuclear staining.

Developmental Neurobiology
Human Fetal Cortical Interneurons 29

forebrain (5–6 gw) migrate tangentially to the PPL, ally seen in layer I. This is in contrast to early fetal
where they are present about a week prior to appear- stages (11–17 weeks) when 35% of CalR+ cells in the
ance of reelin+ Cajal-Retzius cells (Zecevic et al., cortical plate were colabeled with CB (Zecevic et al.,
1999; Meyer et al., 2000). Thus, some of these cells, 1999). Both CalR and CB label neurons in deep CP
including early GABAergic neurons (Zecevic and occasionally resemble pyramidal cells, with triangu-
Milosevic, 1997) probably represent pioneer neurons lar cell bodies and an apical dendrite. This transient
reported previously (Meyer et al., 2000). In following labeling of pyramidal cells by calcium-binding pro-
weeks, CalR+ cells are a part of the emerging CP teins is in accord with previous reports in animal
from the very beginning. They continue to populate models and human fetal cortex (Yan et al., 1997;
cortical layers as they form in the inside-out manner, Zecevic et al., 1999).
migrating radially similar to what has been described By mid-term other subgroups of cortical interneur-
for projection neurons (Rakic, 1974). ons such as Sst+, PV+, and NPY+ cells are detectable,
Tangentially migrating interneurons at early embry- but are sparse and located mainly in the SP. These
onic stages come from the GE, confirming results interneurons become more numerous in humans
reported in rodents (e.g., Anderson et al., 1997; Tama- during the second half of gestation, not studied here.
maki et al., 1997; Parnavelas et al., 2000). The early Indeed, Sst+, PV+, and NPY+ neurons were reported
existence of GABAergic system, even before the emer- in later stages of gestation in human cerebral cortex
gence of the CP, indicates that secreted GABA can (Kostovic et al., 1991; Cao et al., 1996; Delalle et al.,
influence proliferation of cortical progenitor cells from 1997; Yan et al., 1997; Bayatti et al., 2008). Simi-
the very beginning of neurogenesis. Release of GABA larly, in mice these markers were expressed only after
by neurons has differential effect on VZ and SVZ pro- birth (Gonchar et al., 2007).
genitors, promoting their proliferation in the VZ and Consequently we did not observe cells colabeled
inhibiting it in the SVZ (Haydar et al., 2000). Electro- with CalR and either Sst or PV, cell types previously
microscopic studies have shown that the initial synap- reported in mice (Wonders and Anderson, 2006; Xu
ses in the human layer I and subplate appear at around et al., 2006; Miyoshi et al., 2007). In the mouse, 70%
12 gw (Kostovic and Rakic, 1990; Zecevic, 1998). of cortical CalR+ interneurons develop after E14.5,
These results are temporally matched by the expression they do not coexpress either PV or Sst, and they are
of molecular markers of synaptogenesis, such as destined for deep cortical layers. This population of
VGAT (vesicular transporter for GABA) and synpato- CalR+ neurons is bitufted/bipolar, vertically oriented
physin at the same fetal ages (Bayatti et al., 2008). and derived from the CGE (Xu et al., 2004; Butt
Thus, it is likely that spontaneous activity of GABAer- et al., 2005; Wonders and Anderson, 2006). A smaller
gic system could be the initial driving force for cortical CalR+ cell population (30%) can be colabeled with
development (e.g., Ben-Ari, 2002). This is particularly Sst and are derived either from the MGE (Butt et al.,
interesting since at early stages of cortical development 2005; Xu et al., 2006) or dorsal CGE (Xu et al., 2004;
GABA acting through GABAA receptors has an exci- Wonders and Anderson, 2006). They were reported
tatory action, before becoming an inhibitory transmit- to migrate in the inside-out manner and connect with
ter later in life (e.g., Cherubini et al., 1991; Ben-Ari, their targets in all cortical layers (e.g., Xu et al.,
2002; Khazipov and Luhmann, 2006). The switch 2004). Contrasting this view, it has been recently pro-
from excitation to inhibition in GABA action is attrib- posed that cortical interneurons from CGE, regardless
uted to the changing intracellular concentration of of the time of their origin, target pyramidal neurons
chloride [Cl ]i, which depends on the expression of in superficial cortical layers (Miyoshi et al., 2009).
two chloride transporters, NKCC1, expressed early, Interneurons from CGE in the mouse migrate cau-
and KCC2 expressed in more mature neurons. As dally to hippocampus and caudal cerebral cortex
judged by the expression of KCC2, in the human fetal (Nery et al., 2002; Yozu et al., 2005) under COUP-
cortex this switch has started but is not completed by TFII control (Kanatani et al., 2008). In the human
16 gw, when only some neurons in the subplate brain, clear molecular evidence for the existence of
express KCC2 (Bayatti et al., 2008). the well-defined CGE is not yet present.

Cortical Interneurons at Fetal Stages Cortical Origin of a Subtype

+ of Interneurons
Around mid-term, the numbers of both CalR and
CB+ cells increase in the human cerebral cortex. The most intriguing finding in the fetal cerebral
Interestingly, their colocalization was only occasion- cortex is the presence of proliferating CalR+ cells in
Developmental Neurobiology
30 Zecevic et al.

the SVZ at mid-term. Although these cells are a small Subpial Granular Layer As an Additional
percent of all SVZ cells considering the overall size Source of Cortical Interneurons
of the SVZ at mid-term, their total amount may be
quite significant (Yu et al., 2009). Strengthening this Subpial aggregation of cells form a SGL, first
finding, cells expressing Dlx2 and Nkx2.1 transcrip- described as specific for primates (Brun, 1965, Gadis-
tion factors characteristic to the MGE in rodents, are seux et al., 1992, Zecevic and Milosevic, 1997;
numerous in the neocortical SVZ (Rakic and Zecevic, Meyer and Wahle, 1999). Recently, however, this
2003). Moreover, both Nkx2.1+ and Dlx2+ cells pro- layer has been recognized also in rudimentary form
liferate at mid-term at the same neocortical location in rodents (Wichterle et al., 2001). This transient
as CalR+ cells (Jakovcevski et al., in preparation). layer emerges in human forebrain at 11 gw and disap-
These results on fixed fetal cryosections confirm our pears by 27 gw. In primates, SGL is characterized by
earlier findings in the organotypic SVZ slices from the continuous addition of cells for several months. In
human brain that cells containing Nkx2.1 and Dlx2 the monkey, using tritiated thymidine labeling we
take up BrdU, indicating their local proliferation have shown that cells are added to SGL and layer I
(Zecevic et al., 2005). Moreover, on the human fetal for a period of almost 2 months, from E43-93
slices from the SVZ it was demonstrated that retrovi- (Zecevic and Rakic, 2001). Thus, although SGL is
ral labeled cells divide locally several times before not a proliferative zone, it serves as a conduit for neu-
migrating to the upper layers of the cortex (Letinic et rons coming from the olfactory region and region
al., 2002). It has been estimated that 65% of cortical around paleocortical ventricle (Meyer and Wahle,
GABAergic interneurons are derived from the Dlx1/ 1999). SGL disappears relatively quickly at 27 gw,
2+/Mash1+ lineage located in the neocortical VZ/ not only by cell death but probably also by inward
SVZ, whereas 35% have a GE origin (Letinic et al., migration, thus contributing to a subpopulation of
2002). In monkey, early interneuronal progenitors are cortical interneurons (Zecevic and Rakic, 2001;
initially GAD65+/Mash1+ cells in the GE, but later in Rakic and Zecevic, 2003).
gestation another population of GAD65+/Mash1+ In conclusion, several sites of origin for cortical
cells proliferate in the neocortical VZ/SVZ, implying interneurons, including the GE, neocortical VZ/SVZ,
that later born interneurons have cortical origin and the SGL contribute to the complexity of the inter-
(Petanjek et al., 2009). Furthermore, in human cases neuronal population in the human cerebral cortex.
with severe holoprosencephaly, where substantial Importantly, CalR+ cells seem to be a heterogeneous
parts of the GE are missing, only CalR+ neurons were cell population that can be generated at different loca-
demonstrated in the cerebral cortex, while other tions, depending on the stage of development. Early
subgroups of interneurons were absent. This result born CalR+ neurons seem to be derived exclusively
suggests a dorsal origin of cortical CalR+ cells (Fertu- from the GE and assume their final position in deep
zinhos et al., 2009). In addition, we have shown cortical layers. Late generated CalR+ neurons des-
double-labeled GABA+/Nkx2.1+ cells in the embry- tined for superficial layers II/III generated by the end
onic cortical primordium, suggestive of their origin in of corticogenesis appear to have cortical SVZ origin.
the cortical VZ (Rakic and Zecevic, 2003). The The population of calretinin+ cells is spared in the
expansion of the cortical SVZ during evolution has cortex of patients with a severe holoprosencephaly
undoubtedly had a great impact on the evolution of (Fertuzinhos et al., 2009), which implies their cortical
the human cerebral cortex allowing for the increase origin. In rodents dorsally derived interneurons seem
in diversity of neuronal progenitors. At mid-term pro- to be a much smaller population, reported as calreti-
liferation is still very active in the large outer SVZ nin+ cells for olfactory bulbs (Kohwi et al., 2007,
that has no equivalent in rodents (Smart et al., 2002; Yang, 2008), or neonatal GABAergic cells destined
Zecevic et al., 2005; Bayatti et al., 2008). Prolonged for various cortical areas in the mouse (Inta et al.,
proliferation in the primate-specific outer SVZ 2008). Notably, vertically oriented CalR+ cells that
(Lukaszewicz et al., 2006; Dehay and Kennedy, are targeting other interneurons rather than pyramidal
2007), is temporally matched with emergence of neurons are more numerous in upper cortical layers
enlarged supragranular layers (layers II–IV) in prima- of primates relative to rodents (Gabbott et al., 1997,
tes (Hill and Walsh, 2005). These cortical layers are Zaitsev et al., 2005). The predominance of these
essential for forming cortico-cortical connections and slow-spiking CalR+ interneurons in monkey prefron-
development of higher brain functions, such as cogni- tal cortex was suggested to be instrumental for forma-
tive functions, abstract thinking, language, which tion of species specific neocortical circuits important
characterize us as humans (Hill and Walsh, 2005; for cognitive functions of primates (Zaitsev et al.,
Jones, 2009; Rakic, 2009). 2005). In the absence of fate mapping and transgenic
Developmental Neurobiology
Human Fetal Cortical Interneurons 31

approaches in humans, we rely mainly on distribution of Nkx2-1 in the temporal specification of cortical inter-
of immunolabeled cells correlated with specific time neuron subtypes. Neuron 59:722–732.
of development. So far, however, all the studies per- Cao QL, Yan XX, Luo XG, Garey LJ. 1996. Prenatal devel-
formed in primates by different groups using diverse opment of parvalbumin immunoreactivity in the human
striate cortex. Cereb Cortex 6:620–630.
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