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First published online October 26, 2007
doi: 10.1242/10.1242/dev.008524
1 Department of Neurology and Neuroscience, Weill Cornell Medical College, New
York, NY 10065, USA.
2 Department of Psychiatry, College of Physicians and Surgeons, Columbia
University, New York, NY 10032, USA.
3 The New York State Psychiatric Institute, New York, NY 10032, USA.
4 Department of Neurosurgery, Weill Cornell Medical College, New York, NY 10065,
USA.
* Author for correspondence (e-mail: mer2005{at}med.cornell.edu)
Accepted 31 August 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Cyclin D2 (Ccnd2), Parvalbumin interneurons, Cell cycle, Cortical excitability
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In mice, approximately 80% of cortical
interneurons are represented by parvalbumin (PV+), and somatostatin (SSN+)
expressing subtypes that are derived from the medial ganglionic eminence (MGE)
and follow tangential migrations to the cortex
(Xu et al., 2004;
Xu et al., 2003). Although, in
some settings, they may promote neuron firing
(Derchansky et al., 2004), most
GABAergic interneurons are inhibitory, raising the depolarization threshold
for excitatory neurons. Genetic models indicate that loss of
interneuron-mediated GABA transmission can result in lowered seizure
threshold. For example, mice without the synthetic enzyme GAD65 (glutamic acid
decarboxylase 2; also known as Gad2 - Mouse Genome Informatics) lack the
normal postnatal increase in cortical GABA and display paroxysmal
electroencephalographic (EEG) spike activity with increased seizure frequency
(Stork et al., 2000). Genetic
inactivation of uPAR (urokinase plasminogen activator receptor; also known as
Plaur - Mouse Genome Informatics) reduces PV-expressing GABAergic interneurons
in cingulate and parietal cortex as well as SSN-expressing interneurons in the
hippocampus (Eagleson et al.,
2005). Mice lacking uPAR have spontaneous seizures and a lowered
threshold to pharmacologically induced seizures
(Powell et al., 2003).
Deletion of the Dlx1 transcription factor reduces calretinin interneurons,
becoming apparent by postnatal day 60 (P60), while sparing PV+ subtypes
(Cobos et al., 2005).
Dlx1-/- mice reveal generalized spike and spike-wave EEG
patterns in the absence of myoclonic seizures, with higher frequency and
amplitude spike discharges during myoclonic seizures. The uPAR- and
Dlx-null mouse mutants suggest that subtype-specific interneuron
deficits may be distinguishable, and such selective deficits might yield
insight into the function - and perhaps critical developmental emergence - of
particular interneuronal subpopulations.
Mice lacking the G1-active cell cycle protein cyclin D2 (cD2; also known as
Ccnd2 - Mouse Genome Informatics) display a small cerebellum with loss of
granule neurons and virtually no detectable stellate interneurons
(Huard et al., 1999). Despite
these losses, other interneurons, including basket and Golgi cells as well as
Purkinje projection neurons, appear unchanged. cD2-null mice also
exhibit reduced cerebral cortical volume, suggesting that cell deficits might
also be found in the telencephalon. More-recent mapping of cD2 and cyclin D1
(cD1; also known as Ccnd1 - Mouse Genome Informatics) protein expression
during brain development indicates that these two cyclins define separate
progenitor pools in embryonic brain
(Glickstein et al., 2007).
However, the forebrain cytoarchitecture has remained unexplored in
cD2-/- mice.
Here, we have found selective deficits in cortical PV+ interneurons in mice
lacking cD2, associated with reduced GABAergic currents and increased cortical
irritability. Cell cycle data suggest that cD2 exerts stronger suppression on
p27 (also known as p27Kip1 and Cdkn1b - Mouse Genome Informatics) levels than
does cD1, to promote progenitor divisions within the subventricular zone
(SVZ). These SVZ divisions, which have been termed `intermediate progenitor'
or `transit amplifying', appear to be crucial for establishing the proper
density of PV interneurons, but are not crucial for other subtypes, including
SSN neurons, that also derive from the MGE
(Wonders and Anderson,
2006).
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
whereas cyclin D1-nulls deleted exons I, II and III of the cD1 gene
(Sicinski et al., 1995) (both
lines on C57BL/6 background). Breeding pairs (cD2+/-
x cD2+/-) were placed in mating cages at 17.00 and
separated the next morning, designated embryonic day 0 (E0). BAC transgenic
mice (C57BL/6) expressing enhanced green fluorescent protein (eGFP) driven by
the GAD67 (Gad1 - Mouse Genome Informatics) promoter
(Ango et al., 2004) were
interbred with cD2+/- mice to produce
cD2+/+::GAD67eGFP and
cD2-/-::GAD67eGFP offspring.
Immunohistochemistry and immunofluorescence
Immunohistochemical stains for all genotypes were processed in parallel to
control for inter-experiment variability. For adult tissues: brains
transcardially perfused [4% paraformaldehyde (PFA) in 0.1 M phosphate buffer
(PB); for GABA immunofluorescence, 4% PFA with 0.025% glutaraldehyde, pH 7.4]
were postfixed in 4% PFA for 1 hour. For embryonic tissues: embryonic brains
were dissected free and drop-fixed in 4% PFA overnight at 4°C. Adult and
embryonic tissue was processed for paraffin embedding (Tissue Tek 2000, Miles
Laboratories). Brains were sectioned coronally at 4 or 10 µm (embryo and
adult, respectively) and mounted on adhesive-coated slides (Fisher
Scientific), deparaffinized and antigen retrieved in Reveal (Biocare Medical).
Primary antibodies for DAB immunohistochemistry included: anti-somatostatin
(
-SSN; MAB354, Chemicon International; 1:100), anti-parvalbumin
(-PV; 235, Swant; 1:100,000), anti-calbindin (-CB; C9848, Sigma;
1:50,000), anti-calretinin (-CALR; AB1550, Chemicon; 1:5000),
anti-neuropeptide Y (-NPY; Peninsula Laboratories; 1:24,000),
anti-vasoactive intestinal peptide (-VIP; Peninsula; 1:5000),
anti-cyclin D2 (-cD2; AB-4, Lab Vision; 1:1000), anti-cyclin D1
(-cD1; SP4, Lab Vision; 1:5000), anti-Ki67 (-Ki67; SP6, Lab
Vision; 1:1000), anti-BrdU (-BrdU; RPN20EZ, Amersham; 1:50),
anti-phosphohistone H3 (-PH3; 16-189, Upstate Biotechnology; 1:1000),
anti-p27 (-p27; P2092, Sigma; 1:10,000), anti-phosphorylated (ser
807/811) retinoblastoma (-pRb; 9308, Cell Signaling Technologies;
1:200), anti-p57 (1:2000; Novus), anti-Nkx2.1 (Lab Vision; 1:300) and
anti-Mash1 (BD Pharmingen; 1:1000). Sections were incubated in primary
antibody, then in Signet Murine or Rabbit Linking and USA-HRP labeling
reagents (Signet Laboratories). After -SSN or -CALR incubation,
sections were incubated for 30 minutes each in goat anti-rat IgG or rabbit
anti-goat IgG (1:200 in PB containing 0.1% BSA and 0.25% Triton X-100) and
avidin-biotin-peroxidase complex (Vectastain Elite Kit; 1:100 in PB; Vector
Laboratories). Bound immunoperoxidase was visualized with
3,3'-diaminobenzidine in hydrogen peroxide (Signet Laboratories) for 3-6
minutes.
Dual-labeled tissues were incubated in primary antibodies against: (1) GABA
(A2052, Sigma; 1:1000) and PV (Swant; 1:50,000); (2) BrdU (Amersham; 1:50) and
PH3 (Upstate; 1:1000); (3) BrdU (Amersham; 1:50) and Ki67 (Lab Vision;
1:1000); (4) anti-Tuj1 (Tubb3) (Covance; 1:2000) or anti-NeuN (Neuna60)
(Chemicon; 1:4000), and either -cD1 or -cD2 at 4°C for 16
hours. Sections were washed in PB and incubated in Alexa Fluor-conjugated
secondary antibodies (1:500, Invitrogen) for 1 hour, washed, and coverslipped
with Vectashield with DAPI (Vector Laboratories). Sections were photographed
digitally at 4x, 10x and 20x magnification using a SPOT
camera (Diagnostic Instruments).
Immunohistochemistry for stereology
Brains were perfused and postfixed for 1 hour in 4% PFA in PB,
cryoprotected overnight in 30% sucrose (in PB) and sectioned by sliding
microtome (40 µm). One section per 200 µm throughout the forebrain was
immunostained with PV and SSN as described above. In GAD67-eGFP+
mice, sections were incubated in a rabbit anti-GFP (-GFP) antibody
(Molecular Probes; 1:2000) and processed using PV as above.
Quantitative stereology
PV, SSN or GAD67-GFP neuron numbers and density were obtained by
two-dimensional and stereologic counting methods, using a Zeiss (Oberkochen,
Germany) Axioplan2 microscope with internal Z drive fitted with a Ludl XY
motorized stage and digital video camera (MicroBrightField), interfaced with
Stereoinvestigator (MicroBrightField). Interneurons were counted in three
brain regions: the hippocampal formation, somatosensory cortex (S1, barrel
field) and motor cortex (M1, M2). (1) Hippocampus. Because of the non-random
distribution of hippocampal PV+ and SSN+ interneurons, all immunoreactive
interneurons were counted in the entire series of sections (one 40 µm
section per 200 µm) throughout the rostrocaudal extent of the hippocampus.
Laminar and regional boundaries were used as previously defined
(Paxinos and Franklin, 2001).
The total number of pyramidal neurons in the hippocampus was stereologically
estimated and the volume of this lamina, as well as that of the granular layer
of the dentate gyrus, was measured. Optical dissector frames and counting
grids of 30 and 250 µm2 were used. (2) Neocortex. The numbers of
neocortical PV+ and SSN+ interneurons were determined using an unbiased
stereologic method called the optical fractionator
(West et al., 1991).
Neocortical immunolabeled neurons were counted in two subregions within each
area that included superficial (layers II and III) or deep (below layer III)
cortical layers. The numbers of immunoreactive interneurons and of
counterstained neurons, as assessed by morphological criteria that
characterize the neuronal nuclei (Peters
et al., 1991; Vaughan,
1984), were stereologically estimated in the cortex. The barrel
field region of the somatosensory cortex was analyzed in each section of the
series in which it was present, whereas the motor cortex was analyzed in 1 of
every 2 sections in the series. Optical dissector frames and counting grid
sizes of 30 and 250 µm2 or 100 and 200 µm2 were
used to estimate neuronal or immunolabeled interneuron numbers, respectively,
in the somatosensory cortex.
Optical dissector frames and counting grid sizes were chosen to permit
systematic random sampling of three to five neurons within an 8 µm depth
focusing range for each sampling field and more than 200 total neurons for
each subregion within each case. Intra-sample coefficients of error (CE)
(Schmitz and Hof, 2000) were
always less than 0.05 and were equivalent across genotypes. All regions were
sampled at 63x magnification in Koehler illumination conditions. The
volume of the different laminar domains of interest in each subregion was
estimated using the Cavalieri principle. The estimated total interneuron
numbers were expressed as the density per subregion, thereby controlling for
the reduced brain volume in cD2-/- mice. A Student's
t-test was performed (significance P<0.05).
BrdU birthdating
Pregnant dams received BrdU (50 mg/kg) i.p. at E13.5 or E14.5-15.5. Brains
from wild-type and cD2-/- littermates were harvested at
P21, paraffin-embedded and processed to co-immunolabel BrdU and PV or SSN. PV
and SSN neurons that were BrdU-labeled or unlabeled were counted in the
10x field of view of the somatosensory cortex (barrel fields; layers
II-III). The proportion of BrdU+, PV or SSN+ neurons was calculated, averaging
three sections/region for each case, and compared (Student's t-test;
significance P<0.05).
Distribution of mitotic figures and BrdU index
Mice received BrdU (50 mg/kg) i.p. 1 hour prior to brain harvest. To
calculate the proportion of S-phase cells in the ventricular zone (VZ) and
SVZ, PH3 and BrdU double-labeled sections were photographed at 20x and
both channels, in addition to DAPI, were merged in Adobe Photoshop. To
determine the distribution of PH3-labeled M-phase cells, a 100x10 µm
reticule was positioned centrally at the crest of the rostral MGE and PH3 or
all BrdU/DAPI-labeled nuclei were counted. The VZ and SVZ abventricular
boundary was estimated at 75 µm (Bhide,
1996), confirmed by the distribution of PH3+ cells. Three sections
were averaged for each case. The labeling index was the proportion of
DAPI-labeled cells in either region that were BrdU+. The entire MGE was
photographed at 20x and montaged, if necessary, so that the rostral MGE
from the interganglionic sulcus to the preoptic area was visible. The MGE was
divided into dorsal and ventral halves from the midpoint of the crest of the
MGE and the SVZ/VZ division was estimated as above. All labeled nuclei were
counted, averaging three sections for each case. The SVZ/VZ ratio was
calculated. Group means were compared (Student's t-test; significance
P<0.05).
Quiescent (Q) and proliferative (P) fractions
Mice were BrdU-injected (50 mg/kg) at E13.5, for brain harvest at E14.5.
The Q and P fractions were calculated from Ki67 and BrdU dual-labeled 4-µm
paraffin sections, photographed at 20x. BrdU+ cells were counted in
Adobe Photoshop using a 100x10 µm reticule positioned centrally at
the crest of the rostral MGE. The channels were merged and the dual
BrdU+Ki67-labeled nuclei were counted. Three MGE sections were sampled and
averaged for each case. The P fraction was the proportion of all BrdU-labeled
cells that were Ki67-immunoreactive. The Q fraction was the remaining
proportion of BrdU single-labeled cells. Fractions were averaged for each
group and means were compared using a Student's t-test (significance
P<0.05).
Whole-cell voltage clamp recordings
Sex-matched cD2-null and wild-type littermates were recorded in
the same session and the order of recording was counterbalanced across
genotype. Cortical slices were prepared from ketamine/xylazine (90/10
mg/kg)-anesthetized mice, aged 3-4 weeks. The brain was quickly removed into a
cold high-glucose buffer containing 100 mM D-glucose, 75 mM NaCl,
2.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4,
0.7 mM CaCl2, 2 mM MgCl2. Forebrain slices were cut
coronally at 400 µm on a Vibratome (Camden Instruments), then perfused (1
ml/minute) for 30 minutes with an oxygenated (95% O2, 5%
CO2) lactate buffer. For an additional hour, slices were perfused
with oxygenated artificial cerebral spinal fluid (aCSF): 124 mM NaCl, 3 mM
KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 2 mM
CaCl2, 1 mM MgSO4, 10 mM D-glucose. Prior to
recording, each slice was transferred to the recording chamber, maintained at
25 (±1)°C, and perfused with the oxygenated aCSF at 1-3 ml/minute
with a gravity-fed system. Exchange of the bath by gravity occurred within 1
minute, and recordings began no sooner than 5 minutes after exchange of the
perfusion solution.
Whole-cell voltage-clamp recordings were made with borosilicate pipettes
(3-7 M
tip resistance) filled with: 300 mM CsCl (to block potassium
conductances), 1 mM QX-314 Cl- (to block sodium conductances), 2 mM
MgCl2, 0.1 mM CaCl2, 10 mM HEPES, 1 mM EGTA, 2 mM
ATP-Na2, 0.1 mM GTP-Na2, pH 7.3. To isolate miniature
inhibitory postsynaptic currents (mIPSCs) mediated via GABAA
chloride channels, the following drugs were added to the perfusion fluid:
tetrodotoxin (TTX, 1.0 µM) to block impulse-dependent neurotransmitter
release; (±)2-amino-5-phosphonopentanoic acid (50 µM) and
6,7-dinitroquinoxaline-2,3-dione or 6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX, 40 µM) to block fast glutamate-gated conductances. The holding
potential of -70 mV and high internal [Cl-] produced inward
postsynaptic currents (PSCs). Complete blockade of spontaneous PSCs by the
addition of gabazine (20 µM) to the perfusion fluid confirmed their
mediation by GABAA chloride channels. Pyramidal neurons in frontal
or frontoparietal cortex were impaled with pipettes connected via a Ag/AgCl
wire to the headstage of an AxoClamp 2B amplifier (Axon Instruments, Foster
City, CA). Corrections for liquid junction potentials were made offline.
Series resistances were 20-40 M and were compensated offline to avoid
adding excessive baseline noise. Data were collected as Igor Pro (WaveMetrics,
Lake Oswego, OR) `*.wav' 5-second, 1024-bit sweeps. Sweeps
collected over a 2-5 minute period were concatenated and converted to an Axon
Binary File using ABF Utility software (Synaptosoft, Leonia, NJ); MiniAnalysis
software (Synaptosoft) was used to detect inhibitory IPSCs as events having
rise time, amplitude and decay times significantly different from noise
determined from periods in the trace apparently free from mIPSCs. Total mIPSC
frequency and mIPSC amplitude frequency distributions were calculated. The
effect of cD2 genotype on mIPSC frequency was determined using a
Student's independent t-test [(df=11)>4, two-tailed
P<0.001].
Telemetry-based EEG recording
Mice anesthetized with ketamine/xylazine received two electrodes screwed
into the skull and connected via AgCl wire to a transmitter (Data Science
International) implanted under the skin near the shoulder. After 48 hours of
recovery from surgery, mice were housed in conventional polycarbonate cages
placed on top of receivers (Data Science International) that amplified the
signal, which was digitized and stored on a PC running custom-written
MathLab/Visual C++ routines. Recordings were collected for 18 hours. To detect
seizures and count electrographic events, raw EEG data were band-pass filtered
(3-70 Hz). Power spectral EEG analysis detected and quantified differences in
cortical activity between wild-type and cD2-/- siblings
for artifact-free, randomly chosen 10-second epochs. The frequency component
of the fast-fourier transform of each EEG was binned at 25 Hz resolution for
100 epochs of 10 seconds (1000 seconds) and normalized within each animal. The
effect of cD2 genotype on EEG was determined using a parametric
two-tailed t-test.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Sicinski et al., 1995).
cD2-/- mice are also microcephalic (see Fig. S1 in the
supplementary material). As specific interneuron deficits occur in
cD2-/- cerebellum
(Huard et al., 1999), the
present study examined whether specific interneurons are lost in
cD2-/- cortex. Two of the most prevalent interneuron
subtypes, PV- and SSN-expressing, were compared between wild-type (WT) and
cD2-/- cortex (Fig.
1). In cD2-/-, PV neuron numbers were reduced
in neocortex, especially in superficial layers
(Fig. 1A,B), and were also
reduced in the cD2-/- hippocampus
(Fig. 1C,D). Stereologically
determined density measurements of interneuron subtypes in the hippocampus,
somatosensory and motor cortex, allowed for normalization of the impact of
microcephaly. The density of PV interneurons was reduced by 40% in the
cD2-/- hippocampus and by 30% in somatosensory and motor
cortices relative to WT (Fig.
1E). The mean diameter measurements for PV interneurons were
unchanged, indicating no sampling artifact. The SSN cell density in
cD2-/- cortex did not differ from WT
(Fig. 1G). In hippocampus, the
SSN interneuron density was slightly elevated in the stratum oriens of
cD2-/- mice (Fig.
1H-J). The density of all Nissl-labeled neurons, identified by the
appearance of their chromatin, was also stereologically sampled in
somatosensory cortex and there were no differences in packing density in
cD2-/- mice compared with WT (see Fig. S2 in the
supplementary material). The cD2-/- cortex was thinned
overall, but was substantially thinner superficially [42% (layers I-III)
versus 31% (layers IV-VI) reduction from WT], and this was equally true for
cD2- and cD1-null mice. Despite accounting for reduced
volume, the reduction in density of PV interneurons remained more pronounced
in superficial cortical layers (
45-50% reduction in layers I-III versus
20-30% reduction in layers IV-VI, compared with WT). Thus, adult
cD2-/- mice display selective reductions in the PV
interneuron subtype, particularly in superficial layers. Interneuron reductions in cD2-/- may result from defective cell production or survival. PV expression only reaches adult levels in the third postnatal week. PV+ neuron numbers were already reduced at P25 in cD2-/- (see Fig. S3A,B in the supplementary material). Whether decreased PV interneuron density is a general feature of microcephaly was explored. Owing to their diminished lifespan, cD1-/- versus WT littermates were examined at 5-6 weeks of age. Whereas PV neuron density was reduced in the cD2-/- cortex (see Fig. S3C,D in the supplementary material), the pattern in cD1-/- was indistinguishable from WT (see Fig. S3E,F in the supplementary material). cD1-/- mice similarly showed no differences in SSN neuron density or distribution (see Fig. S3G,H in the supplementary material). Microcephalic cD1-/- mice also had thinner superficial cortical layers (see Fig. S2 in the supplementary material). Therefore, the selective PV interneuron deficit in cD2-/- mice is not a general feature of microcephaly or of deficient late-progenitor divisions. There was no sign of increased apoptosis by TUNEL or caspase-3 labeling in the cD2-/- MGE or cortex (see Fig. S4 in the supplementary material). Thus, cD2 loss causes disproportionate deficits in PV interneuron generation.
Reductions in PV immunolabeling reflected cell loss, not simply a decrease
in PV expression (Fig. 2). The
PV subpopulation constitutes approximately 50-60% of all interneurons and,
therefore, the lower GABA neuron density due to decreased PV+ neurons in the
cD2-/- mouse would be predicted to comprise a reduction of
about 15%. Only minor differences in the total GABA+ neuropil and neuronal
number were evident qualitatively in cD2-/- as compared
with WT cortex (Fig. 2A,E).
These differences corresponded to reduced PV immunofluorescence in
cD2 nulls, as compared with WT
(Fig. 2B,F). Most
GABA-immunoreactive cells in the cortex contained PV; however, the number of
GABA-positive PV-negative cortical neurons was not substantially increased in
cD2 nulls (Fig. 2C,G).
Thus, the reductions in PV neuron numbers in cD2-/- mice
are reflected in overall numbers of GABAergic neurons. To further explore the
specificity of this loss, a GAD67 promoter-driven eGFP BAC transgenic
mouse (Ango et al., 2004) was
crossed with cD2+/- mice. In the GAD67-eGFP transgenics, a
subpopulation of medium and large PVergic interneurons can be identified by
their expression of GFP. Numbers of GFP-immunoreactive neurons were reduced in
cD2-/-::GAD67eGFP mice compared with
cD2+/+::GAD67eGFP
(Fig. 2I,J), and the eGFP+
density differences in the cD2-deficient double mutants were identical to the
PV-immunolabeled estimates (see Fig. S5 in the supplementary material).
Therefore, GABAergic interneurons are truly lost in cD2-/-
and deficits are not attributable to an isolated loss of PV expression.
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Reduced synaptic inhibition and increased irritability in the cD2-null cortex
PV+ GABAergic interneurons form inhibitory synapses primarily onto
perisomatic compartments of pyramidal neurons in the hippocampus
(Freund and Buzsaki, 1996) and
neocortex (Kawaguchi and Kubota,
1998; Wang et al.,
2002). In previous studies, the frequency of GABA-mediated mIPSCs
in hippocampal pyramidal neurons correlated with the density of GABAergic
synapses (Hartman et al.,
2006; Swanwick et al.,
2006). Thus, if the decreased density of PV+ GABAergic
interneurons led to a significant decrease in the density of their GABAergic
synapses, we would observe a reduction in mIPSC frequency in cortical
pyramidal neurons. Whole-cell voltage-clamp recordings were used to isolate
and quantify GABAA receptor-mediated mIPSCs in frontal cortical
pyramidal neurons in slices from cD2-/- mice and their
matched WT littermates. Cortical pyramidal neurons exhibited spontaneous
inward currents (Fig. 3A) with
modal rise and decay times of 4 and 18 milliseconds, respectively. The rise
and decay times of these events were consistent with a perisomatic origin of
the events (Swanwick et al.,
2006). The amplitude frequency distribution
(Fig. 3B) showed that the
majority of mIPSCs recorded across all mice had amplitudes of 2-30 pA, with
rarer events of up to 60 pA. These currents were completely blocked by the
presence of the GABAA receptor antagonist gabazine in the bath
(data not shown). The mean frequency of GABAA-mediated mIPSCs was
significantly reduced in cD2 nulls
(Fig. 3C)
(P<0.001). Thus, the reduction in PV+ interneurons in cD2
nulls, as quantified in the anatomical experiments, is associated with a
significant decrease in GABAA-mediated inhibitory synaptic activity
at cortical projection neurons.
The distribution of the mean normalized power spectral density (nPSD) of telemetry-based EEG differed significantly between cD2-/- and WT mice (Fig. 3D-F) (P<0.05). In this example, the cD2-/- animal exhibited higher spectral power over 200-300 Hz, whereas the WT animal exhibited higher spectral power over the 1-100 Hz range. The dominant peak was shifted toward higher frequencies in the cD2-null animal (P<0.05). These results indicate enhanced cortical excitation in nulls compared with WT mice.
|
;
Xu et al., 2004).
Proliferation and cell survival within the MGE were explored in
cD2-/- embryos as potential mechanisms resulting in PV
interneuron deficits.
Cyclins D1 and D2 have distinct expression patterns in the embryonic
forebrain with limited overlap (Glickstein
et al., 2007). cD1 cells are mainly localized to the ventricular
zone (VZ), whereas cD2-labeled cells are more prevalent in the SVZ
(Fig. 4A,B). Whereas cD1
labeling was uniform in the VZ, cD2 appeared more robust in the dorsal lateral
ganglionic eminence (LGE) and ventral MGE, SVZ and VZ. Very few MGE nuclei
co-express cD1 and cD2 (Glickstein et al.,
2007). Each cyclin, therefore, appears to have distinct regional
control of proliferation in the GE. The expression of cD1 does not compensate
for cD2 in the cD2-/- SVZ
(Fig. 4C). By contrast,
cD1-/- mice display cD2-immunolabeling that is enhanced in
the VZ and is present in the SVZ of the entire GE
(Fig. 4D). Thus, cD2
compensates for cD1 loss in the VZ of cD1-/- mice, but cD1
is not induced in the SVZ of the cD2-/- MGE. This
difference in cyclin expression is reflected in the expression of genes
governing interneuron specification, Nkx2.1 and Mash1 (also
known as Ascl1 - Mouse Genome Informatics)
(Fig. 4E-J)
(Marin et al., 2000). Whereas
the area of Nkx2.1 and Mash1 labeling is reduced in the
cD2-/- MGE, their expression is preserved or expanded in
the cD1-/- MGE that induces cD2.
Two G1-S regulatory proteins, the cyclin-dependent kinase inhibitor p27 and
phosphorylated (ser807/811) retinoblastoma (pRb), were examined in the MGE in
cD2-/- and cD1-/-and WT littermates
(Fig. 5). The absence of p57
(also known as p57Kip2 and Cdkn1c - Mouse Genome Informatics) labeling in the
MGE-VZ/SVZ at E12 and E14.5 suggested the selective importance of p27 in the
MGE at these ages (see Fig. S7 in the supplementary material). Compared with
WT embryos, p27 immunoreactivity was elevated in cD2-/-
sections (Fig. 5A,B,E,F),
whereas pRb-immunolabeling was reduced, especially in the
cD2-/- SVZ (Fig.
5D,H). The increased expression of p27 and decreased expression of
pRb in cD2-/- MGE were consistent with slower progression
through G1 phase in the absence of cD2. By contrast, p27 immunoreactivity was
dramatically reduced in the cD1-/- VZ and increased in the
SVZ (Fig. 5J,N), as compared
with either WT littermates (Fig.
5I,M) or cD2 nulls. pRb was somewhat increased in the
cD2-/- VZ, but levels were equivalent in the SVZ of WT
(Fig. 5K,O) and
cD1-/- (Fig.
5L,P) GEs. The overexpression or knockdown of Rb and p27 has
selective effects on cell cycle duration in G1-S phase
(Ferguson and Slack, 2001;
Ferguson et al., 2002;
Mitsuhashi et al., 2001;
Sherr, 2000). Therefore, these
immunolabeling results indicate that cyclins D1 and D2 are poised to
differentially regulate the cell cycle progression, and removal of either
cyclin has distinct effects on MGE proliferation. Moreover, cD2 might have a
specific role in the formation of the SVZ and in the proliferation of its
progenitors.
Proliferation within the VZ or SVZ and cell cycle exit were compared in cD2-/-, cD1-/- and WT MGE. In embryos lacking cD2, PH3 M-phase nuclei had a similar distribution in the GE and this was abbreviated in depth in the SVZ (Fig. 6B,F). Ki67 labeling (S-G2-M-phase cells) in the VZ was reduced in its extension into the SVZ in cD2 nulls (Fig. 6C,D,G,H). Thus, the proliferating progenitor pool was diminished in the cD2-/- SVZ, or the G1 phase was significantly prolonged. By contrast, PH3 and Ki67 staining were indistinguishable between cD1-/- and WT littermates (Fig. 6I-L), indicating that normal density but reduced total numbers of interneurons in these mutants most likely results from fewer overall cell divisions or a survival defect that uniformly affects the MGE. TUNEL and activated caspase-3 immunolabeling revealed no differences between WT and cD2-/- GE at E12, E14.5, or in cortex at E17, P3, P7 or P14, refuting apoptosis in the cD2-/- GE as a mechanism contributing to interneuron deficits (see Fig. S4 in the supplementary material).
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Four striking differences between the cD2 and cD1 knockouts emerge (Table 2). First, cD2 is significantly induced in the VZ of cD1 nulls, whereas cD1 is only marginally increased, if at all, in the SVZ of cD2 nulls. Second, cD2 induction in the cD1-/- VZ is accompanied by a marked suppression of p27 in the VZ, whereas p27 is upregulated in the SVZ of cD2 nulls. Third, phosphorylated Rb is substantially decreased in the SVZ of cD2-null embryos. Fourth, this apparent increase in p27 and decrease in pRb in the cD2-null SVZ are together consistent with the increased Q fraction (proportion of cells exiting the cell cycle) over a 24-hour period in cD2-/- embryos. These results indicate significant distinctions between the regulation of cD2 and cD1 in developing brain and in how these two cyclins balance the proliferation of different progenitor pools.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Xu et al., 2004;
Xu et al., 2003). Although
loss of either cD2 or cD1 results in smaller forebrains, only cD2
nulls have disproportionately reduced neuronal subtypes in cerebellum
(Huard et al., 1999), cortex
and hippocampus (present report). Therefore, cD2 has an indispensable role in
generating the normal proportion of particular neuronal subsets in developing
brain.
|
). In
contrast to interneuron deficits in cD2 nulls, interneuron losses in
the Dlx1 model have been attributed to a survival defect in the
adult. Mice lacking uPAR have deficits in PV interneurons in the adult (but
not juvenile) cerebral cortex and in SSN interneurons in hippocampus and they
also suffer spontaneous seizures (Eagleson
et al., 2005). In mice lacking the tailless gene (Tlx;
now known as Nr2e1 - Mouse Genome Informatics), the numbers of CALR
and SSN interneurons are reduced, whereas the PV subtype is spared.
Tlx mutants have reduced fear- and anxiety-related behavior; a
seizure phenotype has not been reported
(Roy et al., 2002). The
interneuron deficit in cD2-null mice is unique in that it involves
only PV+ interneurons, both in cortex and hippocampus. Moreover, PV
interneuron deficits are detectable in cD2-/- mice at the
earliest ages in which this interneuron phenotype has matured in cortex (3
weeks). Electrophysiological data presented here begin to characterize the
functional consequences of interneuron deficits in cD2-/-
cortex. Cortical projection neurons exhibit a decrease in the frequency of
mIPSCs that are characteristic of perisomatic inhibitory synapses, a large
proportion of which are PVergic (Kawaguchi
and Kubota, 1998). Thus, the decreased density in PV interneurons
in the cD2 nulls has a significant impact on cortical inhibition.
This is further evidenced by the increased frequency and amplitude of
paroxysmal cortical discharges in the EEG of the awake-behaving
cD2-null mouse. In addition, the shift in the dominant spectral power
density peak toward higher frequencies in baseline recordings from
cD2 nulls is consistent with a loss of GABAergic tone or GABA content
of the extracellular milieu resulting in the generation of ictal
discharges.
Cell cycle deregulation as a mechanism contributing to PV interneuron deficits
Reduced PV interneuron numbers may result from aberrant regulation of the
cell cycle leading to reductions in proliferation in the SVZ and premature
terminal differentiation of progenitor cells. Birthdating data presented here
indicate that the proportion of PV (but not SSN) interneurons produced by the
MGE is affected in cD2-/- brains throughout neurogenesis,
but PV production deficits become more severe at later ages as the progenitor
pool is depleted. That normal densities of other interneuron subtypes (e.g.
CALR, VIP) are produced in cD2-/- mice suggests an
MGE-specific requirement for cD2 in the genesis of PV interneurons.
The most direct model for cD2 involvement in histogenesis, supported by
analyses here, is that this cyclin is both differently regulated and has a
different impact on the cell cycle than cD1 in the MGE. Cyclin D1/D2 effects
on the cell cycle are complex and neither is strictly required for cell cycle
progression (Kozar et al.,
2004; Sherr, 2000;
Sherr and Roberts, 2004). They
are non-catalytic activating subunits of cyclin-dependent kinases 4 and 6
(Cdk4/6) that promote the phosphorylation of Rb, which in turn removes Rb
suppression to promote cell cycle progression and induce the E2F family of
transcription factors (Ohnuma and Harris,
2003; Ross, 1996).
Progenitor exit from the cell cycle requires reaching a threshold set by
levels of cyclin D, opposed by levels of Cdk-inhibitors (CdkIs) including
CIP/KIP-family CdkIs p21 (Cdkn1a), p27, p57 and INK-family members including
p18 (Cdkn2c) and p19 (Cdkn2d). Among CdkIs, p27 inhibits cyclin E-Cdk2 action
required for entry into S phase and p27 has a significant influence on brain
size (Geng et al., 2001;
Kiyokawa et al., 1996;
Knoepfler et al., 2002).
Inducible overexpression of p27 in embryonic mouse neural progenitors
lengthens G1 specifically (Mitsuhashi et
al., 2001). G1 cyclins D2 and D1 form complexes with CIP/KIP CdkIs
including p27 and so oppose their cell cycle inhibitory action
(Sherr, 2000). Indeed, genetic
crosses between cD1- and p27-knockout mice demonstrate that
reduced p27 compensates for lost cD1 (Geng
et al., 2001). In addition, p27 has been implicated in the
regulation of transit-amplifying divisions in adult neurogenesis
(Doetsch et al., 2002), which
are divisions most similar to those in the embryonic SVZ
(Noctor et al., 2004).
Phosphorylated Rb is also affected by cyclin loss and is another key component
of terminal mitosis and differentiation of neurons
(Ferguson and Slack, 2001;
Ferguson et al., 2002). The
present study demonstrates that the effects of cD1 and cD2 on the cell cycle
within developing brain are not completely equivalent and their different
roles in brain histogenesis may hinge on their distinct effects on p27 and
pRb.
How might these cell cycle relationships be understood in the context of
the embryonic proliferative ventricular epithelium (PVE)? Recent studies of
embryonic PVE behavior indicate that the VZ and SVZ represent two distinct
niches of progenitor divisions in which the SVZ supports proliferation of an
intermediate progenitor similar to the transit-amplifying divisions of adult
neurogenesis in the SVZ and hippocampus
(Doetsch et al., 1999;
Noctor et al., 2004;
Seri et al., 2001). The
present study supports a model in which cD2 is required to promote the
intermediate progenitor divisions in the SVZ and that these transit-amplifying
divisions are necessary to generate the normal complement of PV-expressing
interneurons in cortex and hippocampus
(Fig. 8B-D). In this model, cD2
suppresses p27 expression and promotes phosphorylation of Rb. In the WT MGE
(Fig. 8B), cD2 is induced in
cells entering the SVZ, where suppression of p27 and promotion of pRb favor
intermediate progenitor proliferation and an appropriate balance of SSN+ and
PV+ interneurons. When cD2 is absent (Fig.
8C), p27 is unopposed and reduced levels of pRb hinder
transit-amplifying divisions, resulting in a reduced density of PV+
interneurons, but a normal density of SSN+ interneurons (although the absolute
numbers of subtype cells are reduced). By contrast, loss of cD1
(Fig. 8D) induces cD2 in the
cD1-/- VZ, which suppresses p27 expression and promotes
pRb levels in the VZ, while cD2 in the SVZ continues to support intermediate
progenitor divisions. Although these cD2-supported divisions may progress more
slowly (Glickstein et al.,
2007), the normal balance between progenitor and intermediate
progenitor divisions is maintained, resulting in normal densities of SSN+ and
PV+ interneurons in cD1 nulls.
Further studies will determine how cD2 can suppress p27 - whether through transcriptional or post-transcriptional events. The data here do not address whether cD2 expression influences not only proliferation, but also the gene expression routines that specify interneuron subtypes (and that are only partially delineated). Nevertheless, the present studies illuminate differences in the impact of cD2 and cD1 on progenitor division and underscore the important contribution of cell cycle control to regional patterning in the developing brain.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/22/4083/DC1
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ACKNOWLEDGMENTS |
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-aminobutyric acid (GABA) and PV in
somatosensory cortex. (A-D) Adult WT and (E-H)
cD2-/- mice. Sections, 10 µm. GABA+ (A,E) and PV+ (B,F)
neurons and processes are distributed in all layers of the cortex and
GABA-immunolabeled neuropil is moderately reduced in
cD2-/- compared with WT. GABA and PV overlay images (C,G)
show that most GABA+ cells in the cortex contain PV; however,
GABA-immunolabeled, PV-negative cortical neurons are not substantially
increased in cD2-/-. (D,H) The GABA- and PV-immunolabeled
neuropil are most substantially reduced in the superficial cortical lamina.
(I,J) In 40 µm sections, GFP-immunoreactive neuron numbers
are reduced in cD2-/-::GAD67eGFP mice compared
with cD2+/+::GAD67eGFP (see Fig. S5 in the
