3. Results
3.8 Conditional CRD-NRG1 overexpression has no effect on interneuronal migration and
NRG1 signaling via ErbB4 has been implicated in the migration of cortical interneurons (Flames et al., 2004). Interneuron numbers and cortical layering are normal in Emx-Cre*Nrg1f/f mice (Fig. 8B-E), but mildly altered in Nrg1-tg mice (Fig.
9C, E). In addition, the NRG1-ICD has been shown to influence cell survival by regulating genes associated with apoptosis (Bao et al., 2003).
To determine possible effects of chronic CRD-NRG1 overexpression beginning at embryonic and postnatal stages on the migration and maintenance of cortical neurons, a large-scale quantification of neurons in Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice was conducted. Chromogenic immunostainings were performed on coronal paraffin sections (bregma -1.7) of 4 months and 1 year old Stop-Nrg1*NEX-Cre, Stop-Nrg1*CKII-Cre, and parental control mice. Brain sections were stained for the pan neuronal marker NeuN and the interneuronal markers GAD67 and parvalbumin and quantified in the cortex and hippocampus.
A semi-automated quantification method was employed for counting NeuN+ neuron numbers, based on thresholded images, including watershed-based rendering of positive signals (Fig. 20A, C). Quantification of NeuN+ neurons in the cortex of 4 month old Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice showed no differences compared to parental controls, except for a minor difference between Stop-Nrg1*NEX-Cre mice and CKII-Cre controls, which harbored significantly less NeuN+ neurons (*p < 0.05), however, due to small group size (n=3), this finding requires cautious interpretation (Fig. 20A, B). At 1 year of age NEX-Cre controls and Stop-Nrg1*NEX-Cre mice showed significantly reduced numbers of NeuN+ neurons
Fig. 20: NEX-Cre and Stop-Nrg1*NEX-Cre mice have reduced numbers of neurons in the cortex at 1 year of age. (A and C) Chromogenic immunostaining for the pan-neuronal marker NeuN on coronal paraffin sections from 4 months and 1 year old Stop-Nrg1*NEX-Cre, Stop-Nrg1*CKII-Cre and control mice (bregma -1.7), and watershed thresholded images used for semi-autoated quantification of NeuN+ neurons. Scale bars, 50 µm. (B) Semi-automated quantification of 500 µm wide columns of the somato-sensory cortex using watershed thresholded images at 4 months of age revealed a slight decrease in neuronal numbers in CKII-Cre mice compared to Stop-Nrg1*NEX-Cre mice (*p < 0.05, one-way ANOVA;
Bonferroni’s multiple comparison test; n-numbers indicated in the graph). (D) Semi-automated quantification of NeuN+ neurons, as in (B), at 1 year of age revealed a ~20 % reduction of neurons in the cortex of NEX-Cre and Stop-Nrg1*NEX-Cre mice, indicating an effect of NEX haploinsufficiency (*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA; Bonferroni’s multiple comparison test; n-numbers indicated in the graph).
compared to other genotypes (Fig. 20C, D). However, this finding emerged from a combination of slightly increased cortical size and reduced numbers of NeuN+ cells in mice harboring the NEX-Cre allele (data not shown), resulting in a significant reduction in cell density. Individually, the number of NeuN+ cells and cortical size were not significantly different compared to other genotypes. As above, the (modestly) reduced density of NeuN+ neurons was associated with NEX haploinsufficiency in NEX-Cre ‘knock-in’ driver mice, rather than Cre expression per se or HA-NRG1 overexpression, since this effect was not observed in Stop-Nrg1*
Fig. 21: NEX-Cre mice show reduced numbers of GAD67+ interneurons in the cortex and hippocampus at 1 year of age. (A) Chromogenic immunostaining for GAD67 on coronal paraffin sections from 4 months old Stop-Nrg1*NEX-Cre, Stop-Nrg1*CKII-Cre and
control mice (bregma -1.7). Dashed lines indicate quantified areas. Boxes show location of high magnification images. Scale bars, 500 µm (overview), 100 µm (high magnifications). (B) Quantification of GAD67+ interneurons in the cortex and hippocampus of 4 months old Stop-Nrg1*NEX-Cre, Stop-Nrg1*CKII-Cre and controls showed no significant differences in interneuron numbers (One-way ANOVA; Bonferroni’s multiple comparison test; n.s., not significant; n-numbers indicated in the graph). (C and D) A similar analysis of GAD67+ interneurons, as in (A and B), was performed at 1 year of age. (C) Exemplary images of GAD67 immunostainings and quantified areas. Scale bars, 500 µm (overview), 100 µm (high magnifications). (D) Quantification of GAD67+ cells revealed reduced interneuronal numbers in the cortex and hippocampus of NEX-Cre mice. Stop-Nrg1*NEX-Cre mice as well showed a trend to reduced interneuron numbers. (*p < 0.05, **p < 0.01; one-way ANOVA with Bonferroni’s multiple comparison test; n-numbers indicated in the graph).
CKII-Cre or CKII-Cre mice (Fig. 20D). Since the NEX-Cre driver line is a widely used tool in neurobiology, possible neuropathological effects of NEX (NeuroD6) haploinsufficiency must be carefully considered.
Next, interneuronal numbers in the somatosensory cortex and hippocampus (bregma -1.7) were quantified at 4 months and 1 year of age following immunostaining of hippocampus of NEX-Cre controls compared to other genotypes (Fig. 21D). Similar as above, increased cortical and hippocampal size, without concomitant increase in cell numbers (data not shown), resulted in reduced GAD67+ cell density in NEX-Cre controls. In contrast, embryonic (Stop-Nrg1*NEX-Cre) or postnatal (Stop-Nrg1*CKII-Cre) HA-NRG1 overexpression showed no major effect on interneuron density in the cortex or hippocampus (Fig. 21D).
PV+ interneurons present the largest group of ErbB4 expressing interneurons (Neddens and Buonanno, 2009; Vullhorst et al., 2009; Fazzari et al., 2010). To specifically examine these cells, coronal paraffin sections were immunostained for parvalbumin. Similar to the GAD67 analysis, interneuron numbers were manually counted in the hippocampus and cortex (bregma -1.7) of Stop-Nrg1*NEX-Cre, Stop-Nrg1*CKII-Cre, and parental control mice at 4 months and 1 year of age (Fig. 22A, C, dashed lines indicate quantified areas). At 4 months no significant differences in the number of PV+ interneurons were observed in the cortex and hippocampus of Stop-Nrg1*NEX-Cre or Stop-Nrg1*CKII-Cre mice compared to controls (Fig. 22A, B).
Fig. 22: NEX-Cre and Stop-Nrg1*NEX-Cre mice have reduced numbers of PV+ interneurons in the cortex and hippocampus at one year of age. (A) Chromogenic immunostainings for PV on coronal paraffin sections from 4 months old Stop-Nrg1*NEX-Cre,
Stop-Nrg1*CKII-Cre and control mice (bregma -1.7). Dashed lines indicate quantified areas.
Boxes highlight positions of high magnification images. Scale bars, 500 µm (overview), 100 µm (high magnifications). (B) Quantification of PV+ interneurons in the cortex and hippocampus at 4 months of age showed no difference between genotypes. (One-way ANOVA; Bonferroni’s multiple comparison test; n.s., not significant; n-numbers indicated in the graph). (C) Similar chromogenic immunostaining for PV as in (A) for 1 year old mice. observed in Stop-Nrg1*NEX-Cre mice, arguing for an effect of NEX haploinsufficiency. Note that in the cortex of 1 year old mice only a 500 µm wide column of the somato-sensory cortex was quantified. (*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test; n-numbers indicated in the graph).
At 1 year of age a significantly reduced density of PV+ interneurons was detected in NEX-Cre controls and Stop-Nrg1*NEX-Cre mice (Fig. 22C, D). As above, a combination of increased cortical and hippocampal sizes and mildly reduced numbers of PV+ interneurons (data not shown) resulted in a significant reduction of cell density. Again, this effect was associated with NEX haploinsufficiency, as it was also present in Stop-Nrg1*NEX-Cre mice, but not CamKII-Cre driver mice (Fig. 22D).
Taken together, HA-NRG1 overexpression in cortical projection neurons (with early embryonic or postnatal onset) caused no major abnormalities in cortical interneuron migration and/or maintenance.
At 1 year of age a significantly reduced density of PV+ interneurons was detected in NEX-Cre controls and Stop-Nrg1*NEX-Cre mice (Fig. 22C, D). As above, a combination of increased cortical and hippocampal sizes and mildly reduced numbers of PV+ interneurons (data not shown) resulted in a significant reduction of cell density. Again, this effect was associated with NEX haploinsufficiency, as it was also present in Stop-Nrg1*NEX-Cre mice, but not CamKII-Cre driver mice (Fig. 22D).
Taken together, HA-NRG1 overexpression in cortical projection neurons (with early embryonic or postnatal onset) caused no major abnormalities in cortical interneuron migration and/or maintenance.