Neuregulin signaling is dispensable for NMDA- and GABA(A)-receptor expression in the cerebellum in vivo

Abstract

Neuregulin-1s (NRG-1s) are a family of growth and differentiation factors with multiple roles in the development and function in different organs including the nervous system. Among the proposed functions of NRG-1s in the nervous system is the regulation of genes encoding certain neurotransmitter receptors during synapse formation as well as of other aspects of synaptic function. Here, we have examined, in granule cells of the cerebellum in vivo, the role of NRGs in the induction of NMDA receptor (NMDA-R) and GABA(A) receptor (GABA(A)-R), which are thought to be induced by NRG-1 secreted by the synaptic inputs. To this end, we used the Cre/loxP system to genetically ablate the NRG receptors ErbB2 and ErbB4 selectively in these cells, thus eliminating all NRG-mediated signaling to them. Unlike previous reports using cultured granule cells to address the same question, we found that the developmental expression patterns of the mRNAs encoding the NR2C subunit of the NMDA-R and the beta2-subunit of the GABA(A)-R is normal in mice lacking the NRG receptors ErbB2 and ErbB4. Likewise, no alterations in cerebellar morphology nor in certain aspects of cerebellar wiring were resolved in these mutants. We conclude that NRG/ErbB signaling to the granule cells is dispensable for the normal development of their synaptic inputs.

Figure 1.

Expression of Cre recombinase and inactivation of erbb2/erbb4 genes in BACa6Cre-C;erbb2fl/fl;erbb4fl/fl mice. A, Increasing levels of Cre mRNA expression in cerebellum during postnatal development with a maximum level in the adult. Quantitative analysis by qRT-PCR. B, Southern blot demonstrating Cre-mediated recombination of the erbb2 and erbb4 floxed allele with a maximum amount of recombination in the adult. C, Expression of Cre recombinase and Cre-driven recombination are largely confined to granule cells as revealed in parasagittal section from adult mutant cerebellum. Comparison of Cre-expressing cells and of DAPI stain reveals Cre expression in >95% of cells in internal granule layer. Most of the DAPI-stained Cre-negative nuclei at the outer margin of the granular layer are likely to represent somata of Purkinje cells, of Golgi cells, and of Bergmann glial cells, which are all located at the interface of the granular layer and the Purkinje cell layer. D, Western blots from adult cerebella of erbb2/erbb4-deficient mice show substantial abolishment of ErbB4, but little change in ErbB2 protein (see Materials and Methods). Right panel, ErbB4, but not ErbB2 in mutant cerebellum, is reduced at P16.

Figure 2.

A, Expression of NR2C mRNA in postnatal development in both wt and erbb2/erbb4 dKO mice are similar. All values are normalized to ribosomal protein L8 (rL8) expression and expressed as fold change relative to P1 wt. All values are from triplicate measurements. Similar results were found in three independent experiments. B, Same for GABA_A-R β2 subunit mRNA.

Figure 3.

Histological stainings of wild-type (A, C, E, G) and erbb2/erbb4-deficient (B, D, F, H) cerebellum. Scale bar, 50 μm. A, B, Cresyl violet staining of wild-type (A) and erbb2/erbb4-deficient (B) cerebellum. Purkinje cells formed a continuous monolayer, and the granule cell and molecular layer were of normal thickness. C, D, Double immunofluorescence for calbindin (red) to label Purkinje cells and for NeuN (green) to label granule cells. No difference can be seen between control (C) and erbb2/erbb4-deficient (D) cerebellum. E, F, Staining for GFAP revealed an arrangement of Bergmann glia cell processes in the molecular layer, which is not distinguishable in both genotypes. Bright staining on top of the molecular layer in (E) represents staining in the meninges (removed in F). G, H, Staining for vGlut2 to reveal synaptic glomeruli in the granule cell layer and climbing fibers in the molecular layer. No difference was detected between wild-type (G) and erbb2/erbb4-deficient (H) cerebellum. Bright staining on top of the molecular layer in (E) represents staining in the meninges (removed in F).

Figure 4.

Absence of multiple climbing fiber innervation, normal granule cell morphology, and NR2C expression in erbb2/erbb4 dKO mice. A, Composite fluorescence image of a Purkinje neuron with the schematic position of two stimulating electrodes (a and b) and a dendritic region used for calcium measurements. Three intensities of stimulation (0, 1, and 2) are illustrated in the relative graph in the bottom; stim 0 below CF activation threshold; stim 1 above CF activation threshold; stim 2 twice stim 1. B, Somatic recordings after CF stimulation with electrodes a and b and with concomitant stimulation of a and b. C, Somatic recordings (left) and associated Δ[Ca²⁺]_i signals (right) after CF stimulation at the three different stimulation intensities for electrodes a and b and by concomitant stimulation of a and b at intensities 1 and 2. D, Mean ± SD of EPSP (white bars) and Δ[Ca²⁺]_i signals (gray bars) normalized to their means obtained from three cells. The EPSP amplitude was estimated as the average over 25 ms after the peak of the second complex spike (see B). E, Top, Reconstruction of a cerebellar granule cell from a P16 wt animal (top) and MF–EPSCs from another wt cerebellar granule cell at P16 (bottom) in control condition (with 10 μm bicuculline), after addition of 10 μm NBQX, and after further addition of 50 μm AP5. F, Same as E but from KO cerebellar granule cells at P16. G, NMDA component of the MF–EPSC averaged over five wt cerebellar granule cells at P16. H, NMDA component of the MF–EPSC averaged over seven KO cerebellar granule cells at P16.

Figure 5.

NR2C in P7/P8 (A) and P3/P4 (B) wt cerebellar slice cultures as fold change relative to that at day 0 in vitro (DIV0). A, With TTX (2 μm) or APV (100 μm) treatment there was a reduction in the NR2C expression. Note that the expression pattern was independent of the presence of serum in the medium. B, No significant differences in NR2C expression were observed in cerebellar cultures treated with NRG1β (5 nm), ErbB4 blocker AG1478 (5 μm), ErbB2 blocker AG879 (5 μm), or with a combination of both blockers AG1478 and AG879. Note that NR2C expression was independent of whether cultures were started at P7/P8 or at P3/P4, i.e., before onset of NR2C expression.