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Neurogenesis and Stereological
Morphometry of Calretinin-
Immunoreactive GABAergic
Interneurons of the Neostriatum
VLADIMIR V. RYMAR, RACHEL SASSEVILLE, KELVIN C. LUK,
AND ABBAS F. SADIKOT*
Department of Neurology and Neurosurgery, Montreal Neurological Institute,
McGill University, Montreal, Quebec H3A 2B4, Canada
ABSTRACT
We determined the neurogenesis characteristics of a distinct subclass of rat striatum
␥-aminobutyric acidergic (GABAergic) interneurons expressing the calcium-binding protein cal-
retinin (CR). Timed-pregnant rats were given an intraperitoneal injection of 5-bromo-2⬘-
deoxyuridine (BrdU), a marker of cell proliferation, on designated days between embryonic day
12 (E12) and E21. CR-immunoreactive (-IR) neurons and BrdU-positive nuclei were labeled in
the adult neostriatum by double immunohistochemistry, and the proportion of double-labeled
cells was quantified. CR-IR interneurons of the neostriatum show maximum birth rates (⬎10%
double labeling) between E14 and E17, with a peak at E15. CR-IR interneurons occupying the
lateral half of the neostriatum become postmitotic prior to medial neurons. In the precomissural
neostriatum, the earliest-born neurons occupy the lateral quadrants and the latest-born neurons
occupy the dorsomedial sector. No significant rostrocaudal neurogenesis gradient is observed.
CR-IR neurons make up 0.5% of the striatal population and are localized in both the patch and
the matrix compartments. CR-IR neurons of the patch compartment are born early (E13–15),
with later-born neurons (E16 –18) populating mainly the matrix compartment. CR-IR cells of the
neostriatum are a distinct subclass of interneurons that are born at an intermediate time during
striatal development and share common neurogenesis characteristics with other interneurons
and projection neurons produced in the ventral telencephalon. J. Comp. Neurol. 469:325–339,
2004. ©2004 Wiley-Liss, Inc.
Indexing terms: basal ganglia; caudate-putamen; bromodeoxyuridine; calcium-binding proteins;
development; patch-matrix
The neostriatum receives massive glutamatergic inputs
from the cerebral cortex and thalamus and monoaminer-
gic afferents from the brainstem (Parent, 1996; Kawagu-
chi, 1997). Striatal efferents to the pallidal segments and
to the midbrain arise from medium-sized ␥-aminobutyric
acidergic (GABAergic) spiny cells, which make up over
90% of striatal neurons (Kemp and Powell, 1971; Grofova,
1975; Somogyi and Smith, 1979). Less numerous aspiny
interneurons receive inputs from striatal afferents and
other striatal neurons. Interneurons play an important
regulatory role in the activity of spiny projection neurons
(Lapper and Bolam, 1992; Lapper et al., 1992; Kawaguchi,
1997; Rudkin and Sadikot, 1999; Sidibe´ and Smith, 1999).
Four largely nonoverlapping classes of striatal interneu-
rons are immunohistochemically identified: 1) large cho-
linergic neurons (Butcher and Hodge, 1976; Bolam et al.,
1984); 2) a class of GABAergic neurons that colocalize
nitric oxide synthase (NOS), somastostatin (SS), and neu-
ropeptide Y (NPY; DiFiglia and Aronin, 1982; Vincent and
Johansson, 1983); 3) GABAergic neurons that contain the
calcium binding protein parvalbumin (PV; Gerfen et al.,
1985; Cowan et al., 1990); 4) and GABAergic neurons that
Grant sponsor: Canadian Institutes of Health Research; Grant number:
MOP53281; Grant sponsor: Parkinson Society of Canada; studentship; Grant
sponsor: March of Dimes Birth Defects Foundation; Grant number: 1FY02-14;
Grant sponsor: Fonds de la recherche en sante´ du Quebec (FRSQ) (V.V.R.);
Grant sponsor: Montreal Neurological Institute Preston-Robb Fellowship
(V.V.R.); Grant sponsor: FRSQ Senior Investigator Award (A.F.S.).
*Correspondence to: Abbas F. Sadikot, Cone Laboratory for Research in
Neurosurgery, Montreal Neurological Institute, Room 109A, 3801 University
St., Montreal, Quebec H3A 2B4, Canada. E-mail: sadikot@bic.mni.mcgill.ca
Received 13 May 2003; Revised 5 September 2003; Accepted 9 September 2003
DOI 10.1002/cne.11008
Published online the week of January 5, 2004 in Wiley InterScience
(www.interscience.wiley.com).
THE JOURNAL OF COMPARATIVE NEUROLOGY 469:325–339 (2004)
©2004 WILEY-LISS, INC.
contain the calcium binding protein calretinin (CR; Jaco-
bowitz and Winsky, 1991; Re´sibois and Rogers, 1992; Ben-
nett and Bolam, 1993; Kubota et al., 1993; Figueredo-
Cardenas et al., 1996; Sadikot et al., 1996).
The neurogenesis timetable of striatal neurons is de-
rived from earlier [
3
H]thymidine autoradiography studies
and more recent studies using an immunohistochemical
method based on nuclear incorporation of the thymidine
analog, 5-bromo-2⬘-deoxyuridine (BrdU). In the rat, stria-
tal projection neurons are born over an extended period
between E13 and E22, with peak neurogenesis at E15–18
(Fentress et al., 1981; Bayer, 1984; Marchand and Lajoie,
1986). Cholinergic interneurons are born early, between
E12 and E15 (Semba et al., 1988; Phelps et al., 1989).
PV-IR (Sadikot and Sasseville, 1997) and SS-IR interneu-
rons (Semba et al., 1988) of the neostriatum are generated
mainly between E14 and E17, with peak neurogenesis at
E15–16. Spatial gradients of neurogenesis can also be
identified. Cholinergic and PV-IR interneurons show
prominent caudorostral gradients of neurogenesis (Semba
et al., 1988; Phelps et al., 1989; Sadikot and Sasseville,
1997). SS-IR interneurons, on the other hand, show no
apparent spatial neurogenesis gradient in the striatum
(Semba et al., 1988). Neurogenesis gradients of neostria-
tum projection neurons are complex and depend on the
rostrocaudal position of neurons in relationship to the
anterior commissure (Bayer, 1984; Marchand and Lajoie,
1986). Neuronal birth date varies with respect to the
chemically heterogeneous patch-matrix compartments of
the striatum (Marchand and Lajoie, 1986; van der Kooy
and Fishell, 1987; Song and Harlan, 1994). Spiny projec-
tion neurons that occupy the patch become postmitotic
early, whereas later-born neurons occupy mainly the ma-
trix compartment. In the case of interneurons, cholinergic
cells destined for patch compartments are born earlier
than those occupying the matrix (van Vulpen and van der
Kooy, 1998). Whether this pattern of distinct neuronal
birth dates within patch-matrix compartments applies to
GABAergic interneurons is unknown.
In the present study, we determined the temporal and
spatial neurogenesis gradients of CR-IR GABAergic neo-
striatal interneurons. We used an immunohistochemical
method based on BrdU incorporation by dividing cells
(Nowakowski et al., 1989), combined with labeling for
chemospecific phenotypic markers in central nervous sys-
tem tissue (del Rio and Soriano, 1989). We have previously
characterized the neurogenesis of striatal PV-IR interneu-
rons with a similar double-labeling method (Sadikot and
Sasseville, 1997). Here we also analyzed the morphology
of CR-IR neurons, including an unbiased estimate of cell
number obtained from stereology. Finally, we character-
ized the distribution of CR-IR with respect to the chemi-
cally heterogeneous striatal patch-matrix compartments
(Graybiel and Ragsdale, 1978; Herkenham and Pert,
1981) and determined differences in birth date of CR-IR
neurons occupying either compartment. This work has
previously been published in abstract form (Rymar and
Sadikot, 2001, 2002).
MATERIALS AND METHODS
Experimental animals
Female rats (Sprague-Dawley, Charles River, LaSalle,
Quebec, Canada) were coupled with males between 4 PM
and 6 PM. The first 24 hr after coupling was designated as
embryonic day 0 (E0). Dams received a single intraperito-
neal (i.p.) injection of BrdU (Sigma, St. Louis, MO; 50
mg/kg, in Tris-buffered saline, pH 7.6) at 5 PM at the onset
of E12–21. Three dams were injected at each designated
day, for a total of 30 animals. All animals were given food
and water ad libitum. Litters were culled to 10 pups per
dam at birth, and animals were weaned at 3 weeks after
birth. Five males were selected from each litter and pro-
cessed for immunohistochemistry between postnatal day
35 (P35) and P42. Seven additional animals of the same
age were used for unbiased stereological estimates of neu-
ronal number and quantification of cell size.
Animals were deeply anesthetized by using an overdose
of sodium pentobarbital (75 mg/kg, i.p.) and perfused trans-
cardially with an initial wash of heparinized 0.9% saline
(50 –100 ml, 4°C), followed by 4% paraformaldehyde in
phosphate buffer (300 ml, 0.1 M, pH 7.4, 4°C). Brains were
immersed for 48 hours in 30% phosphate-buffered sucrose
solution (pH 7.4) and then cut in the coronal plane at 50
m on a sliding freezing microtome. Free-floating sections
were collected in phosphate-buffered saline (PBS; 0.1 M,
pH 7.4) as separate sets so that each set contained every
sixth serial section. Selected adjacent free-floating sec-
tions were processed for double-labeling immunohisto-
chemistry for BrdU and CR, BrdU and calbindin (CB), or
only CR, then mounted on glass slides, dehydrated, and
coverslipped.
Immunohistochemistry
BrdU immunohistochemistry was performed by using
minor modifications of a previously published method (So-
riano and del Rio, 1991; Sadikot and Sasseville, 1997).
Sections were incubated in 0.5% sodium borohydride dis-
solved in PBS for 20 minutes and rinsed twice in PBS.
Next, sections were incubated for 30 minutes in 1% Triton
X-100 in PBS containing 0.03% hydrogen peroxide, fol-
lowed by 1% dimethlysulfoxide (DMSO) in PBS for 10
minutes. Sections were immersed in 2 N HCl in PBS for 60
minutes, then neutralized by rinsing in sodium borate
buffer (0.1 M, pH 8.5) for 5 minutes. After brief washes in
PBS (3 ⫻5 minutes), sections were preincubated in PBS
containing 10% bovine serum albumin (BSA) and 0.3%
Triton X-100 for 30 minutes, briefly rinsed in PBS, and
then incubated for 14 –16 hours in PBS containing mouse
anti-BrdU antibody (1:40; Becton-Dickinson, San Jose,
CA) and 2% BSA (4°C). After three brief rinses in PBS,
sections were incubated in PBS containing secondary an-
tibody (biotinylated antimouse IgG; 1:200; Vector, Burlin-
game, CA) and 2% BSA. After three brief rinses in PBS,
sections were incubated for 1 hour in avidin-biotin com-
plex (ABC; 1%, in PBS; Vector). Next, sections were briefly
rinsed three times in PBS, and the immunohistochemical
reaction product was revealed by incubation for 7–10 min-
utes in a solution containing 0.37 g nickel ammonium
sulfate, 25 mg 3.3⬘-diaminobenzidine tetrahydrochloride
(DAB), and 2 l hydrogen peroxide (30%) dissolved in 100
ml Tris buffer (0.05 M, pH 7.6). This nickel-enhanced
DAB-based chromogen yields a blue-black reaction prod-
uct. Sections were thoroughly rinsed in PBS and then
mounted out of distilled water on glass slides or prepared
for immunohistochemistry for the second antigen.
For double immunohistochemistry, sections were incu-
bated for 14 –16 hours at room temperature in PBS con-
taining a rabbit anti-CR antibody (1:5,000; Swant). After
326 V.V. RYMAR ET AL.
three brief rinses in PBS (3 ⫻5 minutes each), sections
were incubated for 1 hour in PBS containing secondary
antibody (biotinylated anti-rabbit IgG; 1:200; Vector) and
2% BSA. After three rinses in PBS, sections were incu-
bated in ABC (1%, in PBS). A light brown immunohisto-
chemical reaction product was obtained by using a solu-
tion containing 25 mg DAB, 1% imidazole (1.0 M), and 20
l hydrogen peroxide (30%) dissolved in Tris buffer (0.05
M, pH 7.6). After 10 –15 minutes of exposure to the DAB
chromogen, sections were rinsed thoroughly with PBS.
Sections were mounted out of distilled water, air dried,
dehydrated in a graded series of alcohols, cleared in Xy-
lene Substitute (Shandon, Pittsburgh, PA), and cover-
slipped with Permount (Fisher, Fair Lawn, NJ). As control
experiments, we omitted either anti-CR or anti-BrdU from
the immunostaining schedule and noted no evidence of
cross-reactivity. Furthermore, BrdU immunostaining was
clearly nuclear, whereas anti-CR recognized cell bodies
and processes. We also determined the number of striatal
CR-IR neurons in BrdU/CR double-labeled sections and in
sections from the same animals immunostained for CR
only and found similar results. Finally, we used unbiased
stereology to compare the number of CR-IR neurons in
offspring of animals injected with BrdU and in animals
not injected with BrdU, obtaining similar neuron num-
bers, indicating that BrdU itself is not toxic to striatal
CR-IR neurons at the dose used in these experiments.
To determine whether subpopulations of neostriatum
CR-IR neurons were cholinergic, sections from nine ani-
mals were processed for double immunofluorescence for
CR and choline acetyltransferase (ChAT). Free-floating
sections were incubated in TBS (0.1 M Tris, 1.4% NaCl,
pH 7.3) containing primary antibodies against CR (rabbit
anti-CR; 1:5,000; Swant) and ChAT (monoclonal mouse
anti-ChAT; 1:400; Chemicon, Temecula, CA) and 5% nor-
mal goat serum at room temperature (2 hours) and then at
4°C (14 –16 hours). After three brief rinses with TBS, the
sections were incubated with the secondary antibody (Al-
exa 594-conjugated red anti-mouse, 1:600, for anti-ChAT;
Alexa 488-conjugated green anti-rabbit, 1:600, for anti-
CR; Molecular Probes, Eugene, OR) diluted in TBS for 1
hour. After three brief rinses in TBS, sections were
mounted out of distilled water on glass slides and cover-
slipped 30 minutes later with Krystalon 64969 (EM Sci-
ence Harleco). As control experiments, we omitted either
primary antibody from the staining schedule and observed
no evidence of cross-reactivity between anti-CR and anti-
ChAT with immunofluorescence. Sections were analyzed
using a fluorescence microscope (BX-40; Olympus, Tokyo,
Japan) equipped with the appropriate filters. Photomi-
crography was performed with an Optronics microfire dig-
ital camera (model 99808; Optronics, Goleta, CA), and
digital photographs were edited using Adobe Photoshop
5.0 and Adobe Illustrator 10.0 (Adobe Systems Inc., San
Jose, CA).
Quantitative image analysis
of double labeling
At each embryonic age, BrdU-CR double-labeled neu-
rons were analyzed at five comparable coronal striatum
levels selected at 0.75- ⫾0.15-mm intervals, by using the
rat atlas of Paxinos and Watson as a guide (1998; see Fig.
1A). The selection included two postcommissural levels
caudal to the level of decussation of the anterior commis-
sure (equivalent to sections –2.56 and –1.4 mm with re-
spect to the Bregma; Paxinos and Watson, 1998), a level at
which the anterior commissure formed the ventral border
of the neostriatum (equivalent to section – 0.26 mm; Paxi-
nos and Watson, 1998), and two levels rostral to the de-
cussation of the anterior commissure (equivalent to 1.00
mm and 2.2 mm; Paxinos and Watson, 1998). The dorsal,
medial, and lateral limits of the neostriatum were well
defined (Paxinos and Watson, 1998). Ventrally, the neo-
striatum interfaced with the amygdala and substantia
innominata in its postcommissural part and with the nu-
cleus accumbens in its precommissural division. We de-
limited, at the two precommissural levels, the ventral
limit of the neostriatum from the nucleus accumbens with
a line that extended from above the ventralmost part of
the lateral ventricle medially to the tapered external cap-
sule laterally, at an angle of 25° to 30° below the axial
plane (see Fig. 1A,B).
Striatal distribution of single-labeled CR-immunoreactive
(-IR) neurons and double-labeled BrdU-CR neurons was
plotted by using a system for image analysis. The left
hemisphere was used for all quantitative analysis. The
system consists of a light microscope (BX40; Olympus)
equipped with an X-Y movement-sensitive stage (BioPoint
XYZ; LEP, Hawthorne, NY), a Z-axis indicator (MT12
microcator; Heidenhain, Traunreut, Germany), and a
video camera (DC200; DAGE, Michigan City, IN) coupled
to a computer containing software for computer-assisted
image analysis (Stereo Investigator; MicroBrightField
Inc., Colchester, VT). The software allows drawing of out-
lines of striatal sections at low (⫻10 objective) magnifica-
tion and plotting of positions of single (CR)- or double
(BrdU-CR)-labeled neurons evaluated at high (⫻40 objec-
tive) magnification. Photomicrography was performed
with an Optronics Microfire digital camera (model 99808;
Optronics), and digital photographs were edited using
Adobe Photoshop 5.0 and Adobe Illustrator 10.0 (Adobe
Systems Inc.).
Three animals from each BrdU-injected age group were
selected for analysis. Selection was based on clarity of
single- and double-immunohistochemical labeling, least-
color mixing of chromogens, low background staining, and
optimal tissue preservation. The nickel-enhanced DAB
reaction product used as a chromogen for BrdU immuno-
histochemistry revealed blue-black BrdU-containing nu-
clei. In contrast, DAB-labeled CR-IR neuronal cell bodies
were light brown and often displayed staining of associ-
ated dendrites or axons (see Fig. 2A–D). BrdU-IR nuclei of
double-labeled cells took the form of a stippled pattern
(see Fig. 2B), several large coalescent clumps (see Fig. 2C),
or a solid uniform blue-black reaction product (see Fig.
2D). A double-labeled neuron was defined as having at
least three speckles of blue-black BrdU nuclear reaction
product within the brown CR-IR cytoplasm (see Fig. 2B).
Care was taken to exclude instances of false-positive dou-
ble labeling occurring as a result of BrdU-positive nuclei
in close proximity to, but not within, the cytoplasm of
CR-IR neurons. A 3% double-labeling index (BrdU-CR
double-labeled neurons/total CR-IR neurons) was selected
as the limit for “significant” double labeling. A double-
labeling index greater than 10% was referred to as “max-
imal” double labeling (Sadikot and Sasseville, 1997).
Stereology
Unbiased sterelogical estimates of the total number of
neostriatum CR-IR neurons were obtained by applying
327CALRETININ INTERNEURON NEUROGENESIS
the optical fractionator (Gundersen et al., 1988; West et
al., 1996; Luk and Sadikot, 2001; Luk et al., 2003) with
Stereo Investigator. The rostral and caudal limits of the
neostriatum were determined (equivalent to Bregma 2.20
to –3.80 mm; Paxinos and Watson, 1998), and every sixth
serial section of 50 m within this volume was examined.
Typically, 13 coronal sections at 300 m intervals were
analyzed throughout the reference volume (see Fig. 3A).
Mean section thickness after immunohistochemical pro-
cessing, mounting, and coverslipping was 17 m (tissue
shrinkage effect), as measured with a z-axis microcator.
Sampling of the neostriatum was performed by randomly
translating a grid with 300- ⫻300-m squares onto the
section of interest (see Fig. 3B) and applying an optical
dissector consisting of an 80- ⫻80- ⫻10-m brick (see Fig.
3C). Each section contained 10 –121 sampling sites de-
pending on its surface area. Sections were analyzed by
using a ⫻100 lens (oil, numerical aperture of 1.3, with
matching condenser). The optical fractionator was used to
determine the total number of neurons. For analysis of
morphology, a four-ray isotropic nucleator probe (Gun-
dersen et al., 1988) was also applied to estimate the cross-
sectional area and volume of CR-IR interneurons. The
most prominent nucleolus was taken as the unique iden-
tifier in sections counterstained for Nissl substance. The
longest axis of each cell was determined during applica-
tion of the nucleator, and the cells were divided into six
subgroups based on size (see Fig. 3D)
Neurogenesis gradient analysis
To verify lateral-to-medial or a ventral-to-dorsal spatial
neurogenesis gradients, two sections of the precommis-
sural neostriatum (equivalent to Bregma 1.00 mm and
– 0.26 mm; Paxinos and Watson, 1998) were subdivided
geometrically into four quadrants (see Fig. 4B). Counts of
CR-IR neuron labeling and BrdU-CR double labeling
within each of the four quadrants (or lateral and medial
halves) were considered collectively at the two striatal
levels. To determine dorsal-to-ventral gradients, the dor-
sal and ventral halves of sections from either the precom-
missural (equivalent to Bregma 2.20 mm, 1.00 mm) or the
postcommissural (Bregma 1.40 mm, –2.56 mm) striatum
were analyzed.
The double-labeling index of CR-IR neurons was aver-
aged for 3-day intervals (E13–15, E16 –18, E19 –21), and
comparisons were made among the three resulting groups.
Values for the mean double-labeling index in quadrants or
halves were plotted (see Fig. 4B,C). A two-way mixed
ANOVA procedure and Tukey’s HSD post hoc test was
used for statistical analysis. Because the data were
expressed as a percentage, the ANOVA and Tukey’s
HSD tests were repeated with Arc Sin-transformed
data. All main and interaction effects were calculated
with statistical analysis software (SAS 6.12; SAS Insti-
tute Inc, Cary, NC; or Datasim 1.1; Drake Bradley,
Bates College, ME).
Patch-matrix distribution of
CR-IR interneurons
Estimates of CR-IR cell density in the patch, the matrix,
and the “intermediate zone” were made for one section
(equivalent to Bregma 1.00 mm; Paxinos and Watson,
1998) in eight animals. The “intermediate zones” were
defined as annular areas extending 50 m beyond the
patch perimeter (van Vulpen and van der Kooy, 1996).
Patch-matrix boundaries were determined by using CB
immunostains on adjacent sections. Of note, the dorsolat-
eral part of the neostriatum stains poorly with CB. This
region was excluded from patch-matrix analysis. A two-
way mixed ANOVA procedure and Tukey’s HSD post hoc
test were applied to determine statistical differences in
neuronal number and density.
Animals injected with BrdU either between E13 and
E15 (n ⫽6, with two animals injected on each embryonic
day) or during the E16 –18 time interval (n ⫽6, with two
animals injected on each embryonic day) were compared
to determine whether the birth date of CR-IR neurons
differs with respect to different striatal compartments. A
two-way mixed ANOVA procedure and Tukey’s HSD post
hoc test were applied to determine statistical differences
in double-labeling index. When comparing percentages of
double-labeled cells, an additional Arc Sin-transformation
procedure was applied.
RESULTS
Distribution, morphology, and stereological
estimate of total number of neostriatum
CR-IR interneurons
CR-IR interneurons were nonhomogeneously distrib-
uted in the neostriatum (Fig. 1A,B). The most prominent
density gradient was in the rostrocaudal axis, with the
highest concentrations of CR-IR neurons in the rostral
precommissural neostriatum and the lowest concentra-
tions in the caudal striatum (Fig. 1A). The mean number
(⫾SEM, n ⫽30) of CR-IR interneurons at distinct rostro-
caudal coronal levels (Paxinos and Watson, 1998) was
256 ⫾19, 2.20 mm; 209 ⫾12, 1.00 mm; 166 ⫾10, – 0.26
mm; 43 ⫾5, –1.40 mm; 21 ⫾2, –2.56 mm. The medial
aspect of precomissural striatum contained twice as many
neurons as the lateral half (Fig. 1A,B). The highest den-
sity of CR-IR cells was in the dorsomedial quadrant, with
fewer neurons in the dorsolateral and ventromedial quad-
rants and only a light population density in the ventrolat-
eral quadrant. Our observed rostral-to-caudal (Bennett
and Bolam, 1993) and dorsomedial-to-ventrolateral
(Figueredo-Cardenas et al., 1996) gradients are in keeping
with previous observations.
An estimate of the total number of CR-IR interneurons
was determined by unbiased stereology using the optical
fractionator method (Gundersen et al., 1988; West et al.,
1996; Luk and Sadikot, 2001; Luk et al., 2003). The mean
coefficient of error (Gundersen and Jensen, 1987) of CR-IR
neuronal counts was 0.11 ⫾0.005 (n ⫽7). The optical
fractionator method revealed a total of 13,217 ⫾229
(mean ⫾SEM, n ⫽7) CR-IR interneurons in the neostri-
atum contained within a volume of 20.77 ⫾0.58 mm
3
(mean ⫾SEM; n ⫽7). Based on an estimated 2.54 million
(Luk and Sadikot, 2001) to 2.79 million (Oorshot, 1996)
neurons in the neostriatum, our analysis indicated that
CR-IR interneurons represent approximately 0.5% of all
neostriatal neurons in young adult male Sprague-Dawley
rats.
The cell bodies of CR-IR interneurons were mainly me-
dium sized and round, oval, or fusiform, and they varied in
intensity from light to dark brown on DAB immunostains
(Fig. 2). The longest axis of CR-IR cell bodies ranged from
6.2 to 26.5 m (11.3 ⫾0.3 m; mean ⫾SEM; Fig. 3D). The
328 V.V. RYMAR ET AL.
mean cross-sectional area of CR-IR cells ranged from 24 to
235 m
2
(56 ⫾3m
2
; mean ⫾SEM). The average soma
volume ranged from 91 to 3,550 m
3
(368 ⫾23 m
3
;
mean ⫾SEM). Neurons were categorized as small (longest
axis ⱕ7m), medium-sized (7.1–20 m), and large (⬎20
m; Chang et al., 1982; Chang and Kitai, 1982; Kawagu-
chi et al., 1995). Medium-sized cells (7.1–20 m) made up
91% of all CR-IR neurons, with the majority measuring
7.1–15 m. Small (7%; Fig. 2C) or large (⬍2%) neurons
constituted a minority population (Fig. 3D). Small cells
showed a rostrocaudal gradient of decreasing density sim-
ilar to that observed for medium-sized cells. These cells
were preferentially distributed in dorsomedial zones bor-
dering the periphery of the rostral neostriatum, including
the subependymal zones and areas abutting the corpus
callosum and external capsule.
Large CR-IR neurons were comparable in size to cholin-
ergic neurons and were distributed nonhomogeneously in
the neostriatum. These large neurons were preferentially
localized to either the dorsolateral quadrant of the pre-
commissural striatum or the ventral half of the postcom-
missural striatum close to the boundary with the globus
pallidus. Scattered large CR-IR cells were found in the
dorsomedial neostriatum close to the corpus callosum or
subpendymal area. To determine whether these large cells
are also cholinergic, sections were stained by double im-
munofluorescence using antibodies to ChAT and CR. Al-
though double labeling was noted in other forebrain areas,
including the medial septum-diagonal band complex (Fig.
2G,H), there was no instance of double labeling of striatal
CR-IR neurons and cholinergic neurons in the neostria-
tum (Fig. 2E,F). Of note was that the majority of CR-IR
and ChAT-IR neurons in the medial septal-diagonal band
of Broca complex were not doubly labeled, in keeping with
previous studies of this region (Kiss et al., 1997). However,
there were a few instances of double-labeling noted in this
area. For example, in one section (Bregma 0.7 mm), 107
CR-IR and 185 ChAT-IR neurons were counted in the
medial septal-diagonal band complex. We found 13 in-
stances of immunofluorescence double labeling with
CR-IR and ChAT-IR in this region. Most of these double-
labeled cells contained relatively weak CR-IR immunoflu-
Fig. 1. A: Spatial distribution CR-IR interneurons in the neostri-
atum. Plots containing the location of CR-IR single-labeled (⫻) and
BrdU-CR double-labeled (●) interneurons are shown at five coronal
levels through the left striatum (Paxinos and Watson, 1998) in an
adult animal exposed to BrdU in utero at E14. Note the rostral-to-
caudal and medial-to-lateral density gradients of CR-IR neurons.
Orientation and section outlines are also shown (inset). B: Changes
in distribution of double-labeled BrdU-CR neurons during neurogen-
esis. Location of single-labeled (⫻) and double-labeled (●) CR-IR in-
terneurons in adult animals exposed to BrdU in utero at different
time points of neurogenesis at a single coronal level (equivalent to
Bregma 2.20 mm; atlas of Paxinos and Watson, 1998). Note that, early
in neurogenesis, BrdU-CR double-labeled cells predominate in the
lateral striatum, whereas the latest-born interneurons are located
mainly in the dorsomedial quadrant, suggesting a lateral-to-medial
gradient of neurogenesis. Scale bars ⫽500 m.
329CALRETININ INTERNEURON NEUROGENESIS
orescence, except for three cells localized in the horizontal
limb of the diagonal band (Fig. 2G,H).
Timetable and spatial gradients of
neurogenesis of CR-IR interneurons
The double-labeling index of CR-IR interneurons
(BrdU-CR double-labeled cells/total CR-IR cells) in the
neostriatum was “maximal” (⬎10%) between E14 and
E17, with a peak at E15 (Fig. 4A). “Significant” double
labeling (⬎3%) occurred between E13 and E19. CR-IR
interneurons showed no significant neurogenesis gradient
in the rostrocaudal axis (Fig. 5). In contrast, a strong
lateral-to-medial gradient neurogenesis gradient was
present (Fig. 4B). CR-IR neurons in the lateral half of the
striatum became postmitotic at a significantly earlier age
than CR-IR neurons occupying the medial half. In the
E13–15 group, the double-labeling index was significantly
higher in the lateral half compared with the medial half
(ANOVA, Tukey’s HSD test, P⬍0.0001). On the other
hand, in the E16 –18 group, double-labeling index was
significantly higher in the medial part of striatum com-
pared with lateral part (ANOVA, Tukey’s HSD test, P⬍
0.05). The double-labeling index in the medial half of the
striatum in the E19 –21 group remained higher than that
in the lateral part, but this result was not statistically
significant (ANOVA, Tukey’s HSD test, P⬎0.05; Fig. 4B).
There was no detectable ventral-to-dorsal gradient of neu-
rogenesis for CR-IR neurons when the entire rostrocaudal
extent of the striatum was considered collectively. Finally,
there was no statistically significant difference in neuro-
genesis time course of neurons in dorsal or ventral halves
when precommissural or postcommissural levels were an-
alyzed separately. Because data were expressed as a per-
centage, we validated statistical significance using an Arc
Sin transformation and obtained similar results.
To discern further the gradients of neurogenesis in the
precommissural striatum, we compared the double-
labeling index in separate quadrants (Fig. 4C). The neu-
rogenesis time course of CR-IR neurons was virtually
identical in the dorsolateral and ventrolateral quadrants
[Tukey’s HSD test, q (144) ⱕ0.3, P⬎0.05]. These lateral
quadrants contain the earliest-born neurons. Neurons in
the ventrolateral quadrant were born prior to those in the
ventromedial quadrant (Tukey’s HSD test, P⬍0.01). In
turn, neurons in the ventromedial quadrant were born
prior to those in the dorsomedial quadrant (Tukey’s HSD
test, P⬍0.05). When the same test was performed on Arc
Sin-transformed data, significance was marginal, suggest-
ing that the gradient between the two medial quadrants
was minor. As expected, neurons in the dorsolateral quad-
rant were born significantly earlier than those in the
dorsomedial quadrant (Tukey’s HSD test, P⬍0.01). These
data confirm that the strongest neurogenesis gradients
are in the lateral-to-medial axis, with only marginal dor-
Fig. 2. Photomicrographs of BrdU-CR double-labeled interneu-
rons in the striatum (A–D) and examples of analysis to determine
possible CR/ChAT colocalization in the basal forebrain (E–H). A: Two
CR-IR cells, each showing two BrdU-positive stipples. These cells do
not meet the criteria used for double labeling, since they contain fewer
than three BrdU-positive stipples. B–D: Examples of CR-IR cells
counted as doubly labeled, including cells with three BrdU-IR stipples
(B), a coalescent pattern of nuclear staining (C), or solid BrdU-IR
nuclear staining (D). Note the small CR-BrdU cell (⬍7m diameter)
indicated by the arrow (C) alongside a medium-sized double-labeled
cell (10 –15 m category). E–H: Colocalization of CR and ChAT in the
striatum (E,F) or medial septum-diagonal band area (G,H). No exam-
ples of colocalization between CR-IR and ChAT-IR neurons were
observed in the neostriatum (E,F), whereas a few large (⬎20 m)
CR-IR cells colocalized with ChAT-IR in the septum (G,H). Arrows
denote single-labeled CR-IR cells, and arrowheads indicate large cells
doubly labeled for ChAT-IR and CR-IR. Scale bar ⫽10 minD
(applies to A–D); 100 m in E (applies to E–H).
330 V.V. RYMAR ET AL.
soventral differences. In summary, neurons in the lateral
quadrants were born earliest, followed in partially over-
lapping sequence by ventromedial neurons and finally
dorsomedial neurons.
Differential neurogenesis with respect to
patch-matrix compartments
In approximately 60% of the neostriatum, the intensity
of CB immunoreactivity allowed distinction between
patch-matrix compartments. The dorsal and lateral terri-
tory of the neostriatum was poor in CB immunoreactivity,
and patches could not be distinguished in the “sensorimo-
tor” sector based on this marker (Gerfen et al., 1985).
CR-IR neurons were localized in both CB-poor patches
and CB-rich matrix compartments. We noted a higher
density of CR-IR neurons in an “intermediate” or “annu-
lar” zone surrounding the patches (Faull et al., 1989, van
Vulpen and van der Kooy, 1996), within a 50-m distance
from the CB-IR-defined patch boundary (Fig. 6D,E).
The number and density of CR-IR interneurons was
quantified in different striatal compartments (Fig. 6A,B)
in the precommissural striatum (equivalent to Bregma
1.00 mm; Paxinos and Watson, 1998). Most CR-IR neu-
rons were in the matrix compartment. Only 3.6% of CR-IR
interneurons located in areas with well-demonstrated CB-
defined patch-matrix compartments were localized to
patches. The mean (⫾SEM) density of CR-IR cells was
significantly different in the patch compartment (17 ⫾2)
compared with the matrix (27 ⫾2; one-way within
ANOVA, Tukey’s HSD test, P⬍0.05). The intermediate
zone of the matrix had a significantly higher CR-IR cell
density (39 ⫾5) compared with either patches (17 ⫾2;
one-way within ANOVA, Tukey’s HSD test, P⬍0.01) or
the rest of the matrix (24 ⫾2; one-way within ANOVA,
Tukey’s HSD test, P⬍0.05).
We determined whether there was a relationship be-
tween birth date and localization within a specific com-
partment of the striatal mosaic. CR-IR neurons in the
patch compartment are more likely to become postmitotic
during the early time interval (E13–15) compared with
the later time period (E16 –18; two-way mixed ANOVA,
Tukey’s HSD test, P⬍0.001; Fig. 6C). In fact, after E17,
no instances of BrdU-CR double labeling were found in
Fig. 3. Sampling scheme used for stereological quantification of
neostriatum CR-IR interneurons. A: Thirteen coronal sections at reg-
ular intervals spanning the entire striatum were selected for stereo-
logical analysis (adapted from the atlas of Paxinos and Watson, 1998).
B: Sampling sites were located at the intersections of a 300- ⫻300-m
grid placed over the region of interest with the StereoInvestigator
software. C: 80- ⫻80-m counting frames with a depth of 10 m were
applied at each sampling site. Cells in contact with exclusion lines
(dotted) and planes (shaded) were not included in the counting pro-
cedure. D: Histogram showing CR-IR cell size with the longest axis
measurement. Fig. 4. A: Neurogenesis timetable of CR-IR interneurons in the
neostriatum. The combined proportion of BrdU-CR double-labeled
cells in five representative coronal sections (left) was quantified for
animals injected with BrdU on each embryonic day from E12 to E21.
Neurogenesis of CR-IR interneurons spans from E13 to E19 (⬎3%
doubly labeled), with maximal neurogenesis (⬎10% doubly labeled)
between E14 and E17 and a peak at E15. B,C: Histograms illustrating
the BrdU-CR double-labeling index in different halves or quadrants of
the precommissural striatum after in utero exposure to BrdU at
different embryonic ages. B: Early-born interneurons appear predom-
inantly in the lateral striatum; later-born cells are found mainly in
the medial half. C: Analysis of double-labeling index in different
quadrants. No significant differences in the proportion of double-
labeled cells were observed between lateral quadrants. CR-IR neurons
are born first in the lateral quadrants, followed by birth of cells in the
ventromedial quadrant and, finally, the dorsomedial quadrant.
331CALRETININ INTERNEURON NEUROGENESIS
patches. Double-labeling index in the intermediate zone
was not significantly different between the two age inter-
vals. Neurons in the matrix compartment had a greater
tendency to be born at E16 –18, but the difference was not
statistically significant. Finally, the double-labeling index
of patch CR-IR interneurons was significantly higher than
double-labeling index of matrix CR-IR neurons in the
E13–15 group (P⬍0.01), whereas no significant differ-
ence was found in double-labeling index of different com-
partments in the E16 –18 group (P⬎0.05). The data
suggest that CR-IR neurons of the patch compartment are
mainly an early-born population, whereas those of the
intermediate zone and the rest of the matrix are born
during a broader period of neurogenesis.
Patch-matrix compartments were also demonstrated in
animals exposed to BrdU at either earlier (E13,14, or 15)
or later (E18 or 19) dates. Early injections labeled BrdU-
IR-rich patches. Later injections demonstrated a BrdU-
IR-rich matrix and BrdU-IR-poor patches. This form of
patch or matrix labeling in early- or late-BrdU-injected
animals has also been noted in previous studies (van
der Kooy and Fishell, 1987; Song and Harlan, 1994;
Sadikot and Sasseville, 1997). The BrdU/CR double-
immunostained material was used to describe patterns of
arborization of appendages of CR-IR cell bodies in either
patch or matrix compartments (Fig. 7A–D). This type of
analysis requires relatively high spatial resolution and is
therefore best performed in the same section. Some neu-
rons in either patch or matrix compartments showed pro-
cesses that could be followed across boundaries to the
complementary mosaic compartment (Fig. 7A–J). Neurons
with appendages that crossed boundaries were often lo-
cated in the intermediate zone, with processes in some
cases extending to both the patch and the rest of the
matrix compartment (Fig. 7C,D).
DISCUSSION
The main findings of this study are that 1) CR-IR stri-
atal interneurons represent approximately 0.5% of all
neostriatum neurons in young adult male Sprague-
Dawley rats; 2) CR-IR neurons that populate the neostri-
atum go through final mitosis between E13 and E19 (⬎3%
doubly labeled), with maximum neurogenesis (⬎10% dou-
bly labeled) between E14 and E17 and a peak at E15; 3)
there is a prominent lateral-to-medial gradient of neuro-
genesis (rostrocaudal and dorsoventral gradients are
weak or not significant); and 4) CR-IR neurons of the
patch compartment are born early (E13–15). CR-IR neu-
rons that occupy the matrix compartment become postmi-
totic over a broader period of neurogenesis.
Distribution, morphology, and total number
of CR-IR interneurons in the striatum
The striatum contains mainly medium-sized GABAergic
projection neurons and small populations of largely dis-
tinct cholinergic neurons and GABAergic interneuron sub-
types (Kitai et al., 1979; Bolam et al., 1983, 1984). Distinct
subtypes of interneurons containing GABA colocalize the
peptide SS (DiFiglia and Aronin, 1982) or the calcium
binding proteins PV (Gerfen et al., 1985) or CR (Jacobow-
itz and Winsky, 1991; Re´sibois and Rogers, 1992; Bennett
and Bolam, 1993; Kubota et al., 1993; Figueredo-Cardenas
et al., 1996; Sadikot et al., 1996). Unbiased stereology
estimates of total number of neurons in the rodent stria-
tum are derived mainly from work in Sprague-Dawley
(S-D) rats (Oorschot, 1996; West et al., 1996; Luk and
Sadikot, 2001; Luk et al., 2003), with a few studies in
Wistar rats (Larsson et al., 2001) or gerbils (Dam, 1992).
Data are available for the total number of neostriatum
principal neurons (2.79 million, Oorschot, 1996; 2.54 mil-
lion, Luk and Sadikot, 2001), SS-positive (21,300, West et
al., 1996), NPY-positive cells (14,355, Larsson et al.,
2001), PV-positive cells (16,875, Luk and Sadikot, 2001;
16,597, Larsson et al., 2001), and cholinergic interneurons
(6,803, Larsson et al., 2001). SS-positive neurons, there-
fore, make up 0.8% of all neostriatum neurons, whereas
PV-IR and cholinergic neurons make up 0.7% and 0.3% of
the neuronal population, respectively. The present study
suggests that CR-IR neurons account for 0.5% of all neo-
striatum neurons.
These results indicate that interneurons make up ap-
proximately 2.3% of neostriatum neurons, somewhat
lower than estimates based on data from nonstereological
studies (for reviews see Bolam and Bennett, 1993;
Kawaguchi et al., 1995). Despite their small numbers, the
synaptic position and chemical anatomy of interneurons
Fig. 5. Histograms illustrating proportion of double-labeled
BrdU-CR interneurons in five representative coronal sections of the
neostriatum (A–E). Collective comparison of the histograms from
different coronal levels does not reveal a rostrocaudal neurogenesis
gradient.
332 V.V. RYMAR ET AL.
likely allow powerful modulation of the physiology of the
neostriatum (cf. Lapper et al., 1992; Kita, 1993; Rudkin
and Sadikot, 1999; Sidibe´ and Smith, 1999; Koos and
Tepper, 1999; Ramanathan et al., 2002). It is interesting
that the relative ratio of interneurons may differ in ro-
dents compared with primate species. Whereas PV-IR
(Luk and Sadikot, 2001) and SS-IR (West et al., 1996)
neostriatal interneurons outnumber CR-IR interneurons
(present study) in S-D rats, in humans and squirrel mon-
keys neostriatal CR-IR interneurons outnumber PV-IR
and SS-IR interneurons by a ratio of approximately 3:1
(Wu and Parent, 2000). This finding possibly suggests
increased functional importance of CR-IR neurons in pri-
mates compared with other interneuron subtypes.
CR-IR interneurons are distributed throughout the
neostriatum. There is a strong cell density gradient along
Fig. 6. Compartmental distribution of CR-IR interneurons quan-
tified according to their location within the patch (P), intermediate
zone (IZ), or matrix (M) compartments. A,B: Results are shown as
absolute neuron numbers in A or neuronal densities (neurons/mm
2
⫾
SEM) in B. CR-IR neurons are located mainly in the matrix, with
highest density in the intermediate zone. C: Analysis of interneurons
in each compartment according to their birth date revealed that most
early-born neurons were located in patches, whereas matrix neurons
were born over a broader period. D,E: Plots of CR single-labeled (⫻)
and BrdU-CR double-labeled (●) interneurons in neostriatum coronal
sections (Bregma 1.00 mm) from animals exposed in utero to BrdU at
either E13 or E18. The dashed line denotes the boundary between the
calbindin-poor dorsolateral zone and the calbindin-rich area, where
patch-matrix identification is possible. Limits of patches or the inter-
mediate zones are delineated by solid or dotted lines, respectively.
Scale bar ⫽500 m.
333CALRETININ INTERNEURON NEUROGENESIS
the rostrocaudal axis of the neostriatum, in keeping with
previous observations (Bennett and Bolam, 1993). A mod-
erate mediolateral density gradient is also observed in the
precommissural neostriatum. The highest density of
CR-IR interneurons is seen in the dorsomedial quadrant
of the precommissural striatum, with an intermediate
density in the ventromedial quadrant and the lowest den-
sity in lateral quadrants, largely in keeping with previous
work (Figueredo-Cardenas et al., 1996). A dorsolateral
band of striatal tissue, corresponding to an area that la-
bels poorly for CB, contains only scattered CR-IR neurons.
The distribution of CR-IR neurons contrasts sharply
with that of another prominent population of GABAergic
interneurons that expresses the calcium binding protein
PV. PV-IR density is higher in the lateral part of the
precommissural striatum compared with the medial parts
(Gerfen et al., 1985; Celio, 1990; Cowan et al., 1990; Kita
et al., 1990; Kubota and Kawaguchi, 1993; Kubota et al.,
1993; Bennett and Bolam, 1994; Sadikot et al., 1996;
Figueredo-Cardenas et al., 1996; Sadikot and Sasseville,
1997). Comparison of maps of CR-IR (present study) and
PV-IR neurons (Sadikot and Sasseville, 1997) shows that
the distribution gradients in the neostriatum are largely
complementary. Whereas PV-IR neurons are located pref-
erentially in “sensorimotor territories” of the rodent neo-
striatum, CR-IR neurons distribute preferentially in neo-
Fig. 7. Photomicrographs of adjacent BrdU-CR and BrdU-CB
double-immunolabeled sections used for descriptive analysis of mor-
phological distribution of CR-IR neurons with respect to patch-matrix
compartments. In animals exposed to BrdU at early intervals (E13–
15), the boundaries of patches were delineated by BrdU immunostain-
ing. A,B: CR-IR neurons (arrowheads) are noted in a BrdU-rich patch
in an animal exposed to BrdU at E13 (dashed line). C,D: A BrdU-rich
patch in an animal exposed to BrdU at E15 contains a process (arrow)
of a BrdU-CR double-labeled neuron located in the intermediate zone
of the matrix. E–G: Adjacent sections stained for BrdU-CB (E) or
BrdU-CR (F,G) in an animal exposed to BrdU at E16. The dashed line
in E–G denotes the boundaries of a patch identified on the basis of
CB-poor (E) immunostaining. E and F were taken at similar magni-
fication, and G is a high-power view. A BrdU-CR double-labeled
interneuron in a patch (F,G) sends a process (arrow) into the matrix.
H–J: Adjacent sections stained for BrdU-CB (H) or BrdU-CR (I,J) in
an animal exposed to BrdU at E18. The dotted lines in H–J denote
boundaries of the pencil fibers of the striatum, which are identified as
a morphological reference. The CB-rich matrix compartment is shown
in H. The adjacent BrdU-CR-immunostained section at identical mag-
nification (I) and at higher magnification (J) shows a double-labeled
interneuron with processes restricted to the matrix compartment.
Scale bar ⫽50 m in I (applies to A,C,E,F,H,I); 25 m in J (applies to
B,D,G,J).
334 V.V. RYMAR ET AL.
striatum areas that receive afferents mainly from
allocortical-mesocortical areas (Webster, 1961; McGeorge
and Faull, 1989; for review see Berendse et al., 1992). In
primates, these complementary areas correspond broadly
to either the precommissural putamen (sensorimotor ter-
ritory) or the caudate and rostral putamen (associative
territory), respectively (Kemp and Powell, 1970; Kunzle,
1975; Goldman and Nauta, 1977; Yeterian and Van
Hoesen, 1978; Smith and Parent, 1986; for review see
Sadikot et al., 1992a,b).
Consistent with previous studies (Bennett and Bolam,
1993), CR-IR interneurons are mainly oval or round and
less frequently fusiform. Most cell bodies (91%) are
medium-sized (7–20 m, longest axis), and most have cell
diameters between 7.1 and 15 m (82%). In comparison
with other medium-sized interneurons, CR-IR neurons
tend to have smaller soma (SS-IR, 10 –35 m; PV-IR,
15–35 m; Kubota et al., 1993; Kawaguchi et al., 1995;
Figueredo-Cardenas et al., 1996). A few small neurons, ⱕ7
m, and rare large neurons, ⬎20 m, were also identified.
Small or large cells may represent a separate subpopula-
tion or may be part of a continuous distribution of CR-IR
neurons. Some of these small CR-IR cells may belong to
the neuroglioform group identified in previous Golgi im-
pregnation studies (Chang et al., 1982). In the human
striatum, most large CR-IR interneurons colocalize with
cholinergic markers (Cicchetti et al., 1998). In contrast, in
the present study, we demonstrate that large CR-IR neu-
rons do not colocalize with cholinergic markers in the
rodent neostriatum, in keeping with previous publications
(Bennett and Bolam, 1993; Figueredo-Cardenas et al.,
1996). In some studies, a small proportion of CR-IR neu-
rons colocalizes with PV-IR (Kubota et al., 1993;
Figueredo-Cardenas et al., 1996) or SS-IR (Kubota et al.,
1993) neurons, whereas other studies did not reveal any
significant colocalization of CR-IR neurons with either
PV-IR (Bennett and Bolam, 1993) or SS-IR subtypes (Ben-
nett and Bolam, 1993; Figueredo-Cardenas et al., 1996).
Rodent CR-IR neurons are therefore a mainly distinct
population compared with other striatal neurons.
Neurogenesis of CR-IR interneurons
compared with other neurons
of the neostriatum
Striatal projection neurons become postmitotic over a
broad time interval, between E12 and E22–P2 (E0 corre-
sponds to day of fertilization), with maximal mitosis at
E14 –18 (Fentress et al., 1981; Bayer, 1984; Marchand and
Lajoie, 1986). Interneurons have more restricted timeta-
bles of neurogenesis. Striatum cholinergic interneurons
are the earliest born, with the majority of neurogenesis
occurring between E12 and E15 (Bayer 1984; Marchand
and Lajoie, 1986; Semba et al., 1988; Phelps et al., 1989).
SS-IR interneurons are born later, during a narrow time-
table mainly between E15 and E16 (Semba et al., 1988).
PV-IR (Sadikot and Sasseville, 1997) and CR-IR GABAer-
gic interneuron subtypes in the neostriatum have similar
timetables of neurogenesis. Both neuronal subtypes show
significant neurogenesis between E13 and E20, with max-
imal neurogenesis between E14 and E17. Whereas CR-IR
neurogenesis shows a sharp peak at E15, PV-IR neuro-
genesis shows a broader peak at E14 –17.
Significant differences exist in neurogenesis gradients
of striatal subtypes. Projection neurons show a strong
ventrolateral-to-dorsomedial gradient (Fentress et al.,
1981). In the precommissural neostriatum, prominent
“outside-in” (lateral to medial) and caudal-to-rostral neu-
rogenesis gradients are observed (Smart and Sturrock,
1979; Fentress et al., 1981; Bayer 1984; Marchand and
Lajoie, 1986). Exceptionally, in the postcommissural stri-
atum, an “inside-out” and rostral-to-caudal gradient was
observed in one study (Bayer, 1984). In other studies,
caudal-to-rostral and “outside-in” gradients are noted
throughout the neostriatum (Smart and Sturrock, 1979;
Fentress et al., 1981; Marchand and Lajoie, 1986). Neo-
striatal cholinergic interneurons show strong caudal-to-
rostral neurogenesis gradients (Marchand and Lajoie,
1986; Semba et al., 1988; Phelps et al., 1989) and subtle
lateral-to-medial gradients in the precommissural stria-
tum (Semba et al., 1988). PV-IR interneurons show a
similar caudal-to-rostral gradient through the neostria-
tum and outside-in gradients in the precommissural stri-
atum (Sadikot and Sasseville, 1997). SS-IR interneurons
do not show significant spatial gradients of neurogenesis
(Semba et al., 1988), possibly reflecting a heterogeneous
cell population (Rushlow et al., 1996; Sadikot and Sassev-
ille, 1997).
CR-IR interneurons show a prominent lateral-to-medial
gradient, as is the case for projection neurons and PV-IR
interneurons and, to a lesser extent, cholinergic interneu-
rons. In contrast to cholinergic and PV-IR interneurons,
CR-IR neurons show only a weak or absent caudal-to-
rostral gradient. The absence of a detectable caudal-to-
rostral gradient in our material may reflect the sharp
neurogenesis peak seen at E15, possibly masking a gradi-
ent occurring over a short interval. In the precommissural
striatum, the ventrolateral and dorsolateral quadrants
contain the earliest-born CR-IR neurons, followed by the
ventromedial quadrant. The latest-born neurons occupy
the dorsomedial quadrant. Interestingly, projection neu-
rons show remarkably similar ventrolateral-to-dorsomedial
gradients.
The “outside-in” pattern of neurogenesis observed with
most neostriatum neuronal subtypes is also seen in other
regions of the basal forebrain (Creps, 1974; ten Donkelaar
and Dederen, 1979; Bayer and Altman, 1987), suggesting
a general organizing principle of neurogenesis of both
projection neurons and interneurons in the basal telen-
cephalon (Fentress et al., 1981; Bayer and Altman, 1987;
Sadikot and Sasseville, 1997). This contrasts with neuro-
genesis of projection neurons in the cerebral cortex, where
later-born cells migrate past early-born neurons, yielding
an “inside-out” pattern (Angevine and Sidman, 1961;
Berry and Rogers, 1965; Rakic, 1971). Recent studies sug-
gest that GABAergic interneurons that populate the cor-
tex originate from the ventral telencephalon (de Carlos et
al., 1996; Anderson et al., 1997, 2001; Tamamaki et al.,
1997; Lavdas et al., 1999; Wichterle, 1999). It would there-
fore be of interest to determine whether PV-IR and CR-IR
subtypes of cortical GABAergic interneurons show time-
tables and neurogenesis gradients similar to those of their
counterparts in the ventral telencephalon. Our prelimi-
nary work suggests that cortical and striatal CR-IR inter-
neurons show neurogenesis patterns similar to those of
their subcortical counterparts (Sadikot and Rymar, 2000;
Rymar and Sadikot, 2001).
335CALRETININ INTERNEURON NEUROGENESIS
Localization and birth date of CR-IR
interneurons with respect to patch
and matrix compartments
Striatal neurons are disposed to patch and matrix com-
partments that may be distinguished on the basis of chem-
ical anatomy or with respect to organization of afferents
and efferents (for reviews see Tennyson et al., 1972; Gray-
biel and Ragsdale, 1978; Herkenham and Pert, 1981; Ger-
fen, 1992; Sadikot et al 1992b). Cholinergic (Graybiel et
al., 1986; van Vulpen and van der Kooy, 1996), SS-IR
(Gerfen, 1984; Rushlow et al., 1996), and PV-IR (Cowan et
al., 1990) interneurons populate both the patch and the
matrix compartments of the striatum. Our results suggest
that CR-IR interneurons of the neostriatum are nonhomo-
geneously distributed with respect to patch-matrix com-
partments. Calbindin labels the matrix compartment in
most striatal areas, except at a dorsolateral band corre-
sponding to an area that receives sensorimotor afferents.
This sensorimotor zone is also relatively poor in CR-IR
neurons. The calbindin-rich matrix compartment has a
significantly higher density of CR-IR neurons compared
with CB-poor patches. This difference can be partially
attributed to a high concentration of CR-IR interneurons
in the peripatch matrix compartment or intermediate
zone.
Neurons in either patch or matrix compartments (espe-
cially in the intermediate zone) show processes that can be
followed across boundaries to the complementary mosaic
compartment. This distribution supports the hypothesis
that striatal CR-IR interneurons participate in intercom-
partmental communication. Other interneuron subtypes,
including SS-IR (Gerfen, 1984; Rushlow et al., 1996), cho-
linergic (Bolam et al., 1988; Kubota and Kawaguchi,
1993), and PV-IR (Cowan et al., 1990) neurons also send
processes to the complementary mosaic area and may also
serve to allow communication between compartments.
The peripatch intermediate zone also contains a higher
concentration of cholinergic interneurons (van Vulpen and
van der Kooy, 1996). Although processes of most projec-
tion neurons of the striatum respect compartmental
boundaries, a few neurons at the peripatch boundary zone
send processes into the patch compartment and may,
therefore, serve as an additional substrate for patch-
matrix communication (Bolam et al., 1988; Penny et al.,
1988).
In the rat, neurons of the patch compartment are gen-
erated mainly between E12 and E16, whereas the major-
ity of neurons that occupy the matrix compartment are
born between E17 and E20 (Marchand and Lajoie, 1986;
van der Kooy and Fishell, 1987; Fishell and van der Kooy,
1991). CR-IR interneurons in the patch comparment are
born early, whereas neurons in the peripatch compart-
ment and the rest of the matrix are born over a broader
timetable. Striatal cholinergic neurons that populate the
patch compartment are also born earlier than their coun-
terparts in the peripatch intermediate zone and in the rest
of the matrix (van Vulpen and van der Kooy, 1996, 1998).
This finding indicates that compartmental difference in
birth date may be a general property of interneurons,
analogous to differences in birth dates for projection neu-
rons that occupy the patch or matrix. Such differences in
birth date may be due to possible decreased neuronal
adhesiveness as neurogenesis progresses, resulting in
wider dispersion of later-born neurons (Krushel et al.,
1995; van Vulpen and van der Kooy, 1998). Recent work
suggests a role for diffusible molecules, including Slit-1
and Netrin-1, in repelling cells from the ventricular zone
of the striatal ganglionic eminence zone (Zhu et al., 1999;
Hamasaki et al., 2001). Netrin-1 expressed in the ventric-
ular zone may guide later-born matrix neurons into the
striatal primordium (Hamasaki et al., 2001). In addition,
transcription factors of the homedomain and basic helix-
loop-helix (bHLH) gene families may play a role in deter-
mining the patch or matrix location of early- or later-born
neurons. For example, Dlx1/2 and Ebf1 mutants fail to
generate later-born striatal neurons (Anderson et al.,
1997; Garel et al., 1999), whereas Mash1 mutants and
GSH1,2 double mutants show impaired generation of
early-born striatal neurons (Casarosa et al., 1999; Tores-
son and Campbell, 2001). The available data based on
transciption factor mutants do not as yet elucidate mech-
anisms that may guide the mainly early-born GABAergic
interneuron subpopulation to either the patch or the ma-
trix compartment. Further attention to transcription fac-
tors, adhesions molecules, or receptors specifically ex-
pressed by interneurons may help to elucidate
mechanisms by which these subgroups populate different
compartments.
Origin of the striatal interneurons
The proliferative neuroepithelium of the lateral gangli-
onic eminence is the main source of striatal principal
neurons (Pakzaban et al., 1993; Deacon et al., 1994; Ols-
son et al., 1995; Campbell et al., 1995). Recent evidence
suggests that most interneurons of the forebrain, includ-
ing the striatum, arise from the medial ganglionic emi-
nence and neighboring preoptic and anterior entopedun-
cular area (Olsson et al., 1998; Lavdas et al., 1999;
Wichterle et al., 1999; Marin et al., 2000). Studies exam-
ining mutants lacking specific transcription factors (e.g.,
Mash1, NKX2.1) suggest that cholinergic, CR-IR, and
SS-IR interneurons that populate both the striatum and
the cerebral cortex originate mainly in the medial gangli-
onic eminence (Sussel et al., 1999; Marin et al., 2000). In
the case of CR-IR interneurons, the cell loss in Mash1–/–
and NKX2.1–/– mutants is mainly at caudal levels, with
less prominent reduction at rostral striatal levels (Marin
et al., 2000). Our quantitative study suggests that only
15% of CR-IR interneurons are located caudally to the
anterior commissure. This finding leaves open the possi-
bility that some CR-IR interneurons may arise from a
source other than the medial ganglionic eminence.
Because PV expression is minimal in the neonatal fore-
brain, it is not possible to reach a conclusion on the origin
of neurons based on study of the available transcription
factor mutants, which generally die shortly after birth.
The protein product of the NKX2.1 gene is expressed at an
early developmental phase, mainly in the medial gangli-
onic eminence, and is present in most striatal interneu-
rons in adults, including PV-IR cells (Marin et al., 2000).
Furthermore, the time course of neurogenesis for striatal
PV-IR and CR-IR interneurons is remarkably similar
(Sadikot and Sasseville, 1997; present results). Taken to-
gether, these findings support the hypothesis that a sig-
nificant proportion of PV-IR interneurons is also gener-
ated from the medial ganglionic eminence. The present
evidence does not, however, preclude the possibility that
some PV-IR interneurons are generated in the lateral
ganglionic eminence. Our recent work on the neurogenesis
336 V.V. RYMAR ET AL.
period of cortical CR-IR and PV-IR interneurons suggests
a period of neurogenesis similar to that of their striatal
counterparts (Rymar and Sadikot, 2001), in keeping with
the notion that most forebrain interneurons originate in
the ventral telencephalon.
A common origin for interneurons of the forebrain in the
basal telencephalon has important potential implications
for developmental disorders. Events influencing GABAer-
gic or cholinergic neurogenesis at E14 –18 (equivalent to
gestational age 7–12 weeks in humans; Bayer et al., 1993)
would influence the excitability of the entire forebrain
and, therefore, potentially contribute to the pathogenesis
of a wide variety of developmental disorders, including the
cerebral palsies and the epilepsies. We have recently dem-
onstrated that proliferation of GABAergic projection neu-
rons and interneurons originating in the basal telenceph-
alon is dependent on N-methyl-D-aspartate (NMDA)
receptor activation (Sadikot et al., 1998; Luk et al., 2003).
A wide variety of prenatal insults is mediated by NMDA
receptors, including ischemia and toxic exposure to drugs
of abuse (e.g., phencyclidine, ethanol; Tabakoff et al.,
1991; Deutsch et al., 1998), sedatives, anticonvulsants,
and anesthetics (Reich and Silvay, 1989; Jevtovic-
Todorovic et al., 1998; Morrell, 1999). Brain injury as
early as the first trimester of pregnancy may therefore
alter GABAergic cell number and modulate the excitabil-
ity of the forebrain.
CONCLUSIONS
Striatal neuronal subtypes show distinct patterns of
neurogenesis with common features. Cholinergic inter-
neurons are born first, followed by GABAergic interneu-
ron subtypes. Both interneurons and projection neurons
occupying the patch compartment are born early in com-
parison with projection neurons of the matrix. Interneu-
rons occupying the patch compartment are born earlier
than most interneurons of the matrix. With the exception
of SS-IR subtypes, all striatal interneurons show caudal-
to-rostral and lateral-to-medial gradients of neurogenesis.
Increasing evidence suggests that GABAergic interneu-
rons of the entire forebrain originate in the ventral telen-
cephalon. Neurogenesis characteristics of specific striatal
interneuron subpopulations may, therefore, also be rele-
vant to patterns of neurogenesis of homologous neurons
occupying the rest of the forebrain.
ACKNOWLEDGMENTS
The authors thank Marie-Claude Be´langer and Rubina
Rangwala for technical and administrative assistance.
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