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First published online October 26, 2007
doi: 10.1242/10.1242/dev.008524

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Selective cortical interneuron and GABA deficits in cyclin D2-null mice

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.

View larger version (66K): [in this window] [in a new window]   Fig. 1. Parvalbumin (PV) and somatostatin (SSN) immunoreactivities in somatosensory cortex and hippocampus of wild-type and cD2-/- mice. (A,B) PV neurons are especially reduced in superficial cortical layers in cD2-/-. (C,D) Hematoxylin-counterstained sections show PV interneurons are reduced in the cD2-/- hippocampus. (E) PV neuron density is reduced in all regions of cD2-/- cortex (HP>SS1, M1/M2). (F,G) The relative SSN neuron density distribution is similar in WT and cD2-/- nulls despite cortex thinning. (H,I) The density of SSN-immunolabeled neurons is slightly increased in the hippocampal stratum oriens and dentate of cD2-/-. (J) The SSN neuron density is unchanged in cD2-/- mice. (A-D,F-I) 40 µm sections. a, alveus; o, stratum oriens; p, stratum pyramidale; r, stratum radiatum; l-m, lacunosum moleculare layer; dnt, dentate gyrus; HP, hippocampus; SS1(bf), primary somatosensory cortex (barrel field region); M1/M2, primary and secondary motor cortex; WT, wild type. Mean±s.e.m. densities of PV-IR neurons in the null are expressed as percentage of control (WT). n=5-8 per genotype per region; *, P<0.02; **, P<0.001.

View larger version (101K): [in this window] [in a new window]   Fig. 2. Co-localization of -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 supplementary material). Therefore, GABAergic neurons are truly lost in cD2-/- mice.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Consequences of PV-interneuron loss in cD2-/- brain. (A-C) Whole-cell voltage-clamp recordings in acute slices show GABAA receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) in frontal cortical pyramidal neurons. (A) Example traces under conditions isolating GABAA receptor-mediated mIPSCs (see text). (B) Mean frequency distribution of mIPSC amplitudes in cD2 mutant (black) and WT (gray) neurons. (C) Average frequency of mIPSCs of all amplitudes. *, cD2 nulls<WT, P<0.05. (D-F) Cortical electroencephalographic (EEG) recorded by telemetry from awake-behaving mice. (D) Example traces for cyclin cD2-/- and WT siblings show intermittent bursts of faster frequency discharges in the cD2 null. (E) Mean normalized power spectral density (nPSD) distribution over 1000 seconds, collected in randomly selected blocks of 10 seconds each in cD2 mutant (black) and WT (gray) with 25 Hz bin resolution. (F) Average of mean nPSD of EEG for low (1-175 Hz) and high (200-375 Hz) frequency. *, P<0.05, as compared with WT.

View larger version (89K): [in this window] [in a new window]   Fig. 4. Reciprocal cyclin D expression changes in cD1-/- or cD2-/- mice at E14.5. (A-D) The distribution of nuclei expressing cD1 or cD2 proteins is distinct and non-overlapping in the medial ganglionic eminence (MGE) at E14.5. Compared with WT (A,B), mice lacking cD2 (C) have as many cD1-immunoreactive cells in the VZ as WT MGE, but the SVZ has a few more cD1+ cells, partially compensating for loss of cD2. In mice lacking cD1 (D), cD2-immunoreactive nuclei are more prevalent in the VZ and are also observed in the deep SVZ to an extent similar to that in WT. (E-G) Compared with WT (E), Nkx2.1 expression in the E14.5 MGE is reduced in area in cD2-null mice (F), and the same or increased in cD1-null mice (G) (MGE perimeter bordered with dashed line). The asterisk denotes Nkx2.1 labeling in the mantle region that was present in all genotypes but more easily captured here owing to reduced MGE volume. (H-J) Compared with WT (H), Mash1 expression in the MGE is reduced in cD2-null SVZ (I) and enhanced in cD1-null SVZ (J) (dashed line demarcates the VZ/SVZ border).

View larger version (143K): [in this window] [in a new window]   Fig. 5. Cdk-inhibitor p27 and cD-Cdk effector retinoblastoma in the MGE of cD2-/- and cD1-/- mice as compared with wild-type littermates. (A-H) At E12.5 (A-D) and E14.5 (E-H), the labeling-intensity of p27 (A,B,E,F) is increased and fewer nuclei label with phosphorylated Rb (pRb) (C,D,G,H) in cD2-/- mice than in the WT. (I-P) The distribution of p27+ cells is reduced specifically in the VZ of cD1-/- (J,N), as compared with WT (I,M). pRb labeling is equivalent in cD1-/- and WT MGE.

View larger version (124K): [in this window] [in a new window]   Fig. 6. Proliferation markers PH3 and Ki67 are altered in the MGE of cD2-/-, cD1-/- and WT littermates. (A,B,E,F) The numbers of PH3-labeled M-phase nuclei are reduced in both the VZ and SVZ of cD2-/- mice. The distribution of PH3-immunolabeled nuclei is most substantially reduced in cD2-/- in the ventral SVZ. (C,D,G,H) The numbers and distribution of Ki67-immunoreactive nuclei are also reduced in the SVZ of cD2-/- as compared with WT. (I,J) The numbers and distribution of PH3+ nuclei in cD1-/- and WT are identical. (K,L) Distribution of Ki67+ nuclei is similar in WT and cD1-/-. Proliferation in the VZ and SVZ of cD1 nulls is less affected than in cD2-/- mice.

View larger version (49K): [in this window] [in a new window]   Fig. 7. Reduced proliferation and early progenitor cell cycle exit in cD2-/- MGE. All sections, 4 µm. (A) Fewer nuclei label with the M-phase marker PH3 in the VZ and SVZ of the cD2-/- MGE than in WT. (B-D) Fewer nuclei are labeled by a 1-hour BrdU pulse in cD2-/- (C) than WT (D) MGE. The proportion of total (DAPI) nuclei labeled by anti-BrdU is significantly reduced in the SVZ. (E-G) Dual-immunolabeling with BrdU and Ki67 (to label S-phase through M-phase cells) shows the total numbers of nuclei labeled by a 24-hour BrdU pulse is slightly reduced in cD2-/- MGE, but numbers of BrdU+ nuclei not co-labeled with Ki67+ are increased in the MGE of cD2 nulls (F), as compared with WT (E). Quantification of dual (P fraction) and single (Q fraction; BrdU alone) labeled cells indicates a decrease in the proliferating, P fraction and a corresponding increase in the quiescent, Q fraction in cD2-/- MGE (G), suggesting progenitors are prematurely depleted in cD2-/- mice. *, P<0.05; **, P<0.01; ***, P<0.003.

View larger version (20K): [in this window] [in a new window]   Fig. 8. Mechanism of PV loss in the cD2-/- MGE. (A) Deficits in PV interneuron production occur in early and late MGE populations. The proportion of PV+ cells in layer II-III somatosensory cortex that is also labeled by BrdU injected at E13.5 (n=2 per genotype) or E14.5-15.5 (n=5 per genotype) is significantly reduced in cD2-/- mice, whereas the proportion of SSN+ cells generated at any age is the same in cD2-/- and WT littermates. *, P<0.05; **, P<0.002. (B-D) Model of D cyclin roles in the MGE. cD2 normally suppresses p27 levels more strongly than does cD1, requiring more p27 to accumulate before cells can exit the cycle. cD2 also promotes phosphorylation of Rb (pRb), facilitating progression through G1 phase and perhaps influencing downstream gene transcription. cD2 support of SVZ divisions promotes the balanced production of SSN+ and PV+ interneurons. (C) In the absence of cD2, constraints on p27 are lifted and pRb levels fall, hastening cell cycle exit and leaving the proportion of SSN+ neurons unchanged or slightly increased, while disproportionately fewer PV+ interneurons are generated. (D) Loss of cD1 induces cD2 in the VZ, which inhibits p27 and promotes pRb. Divisions continue in both the VZ and SVZ, although they may be slower overall. This leads to fewer rounds of division and reduced total neuronal numbers, but preserved SSN+ and PV+ neuron densities in cD1-/- brains.

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