Bidirectional regulation of Munc13-3 protein expression by age and dark rearing during the critical period in mouse visual cortex

Abstract

Rearing in darkness slows the time course of the visual cortical critical period, such that at 5 weeks of age normal cats are more plastic than dark-reared cats, while at 20 weeks dark-reared cats are more plastic [Mower GD (1991) The effect of dark rearing on the time course of the critical period in cat visual cortex. Dev Brain Res 58:151-158]. Thus, genes that are important for visual cortical plasticity should show differences in expression between normal and dark-reared visual cortex that are of opposite direction in young versus older animals. Previously, we showed by differential display polymerase chain reaction and northern blotting that mRNA for Munc13-3, a mammalian homologue of the C. elegans uncoordinated (unc) gene, shows such bidirectional regulation in cat visual cortex [Yang CB, Zheng YT, Li GY, Mower GD (2002) Identification of Munc13-3 as a candidate gene for critical period neuroplasticity in visual cortex. J Neurosci 22:8614-8618]. Here, the analysis is extended to Munc13-3 protein in mouse visual cortex, which will provide the basis for gene manipulation analysis. In mice, Munc13-3 protein was elevated 2.3-fold in dark-reared compared with normal visual cortex at 3.5 weeks and 2.0-fold in normal compared with dark-reared visual cortex at 9.5 weeks. Analysis of variance of protein levels showed a significant interaction, indicating that the effect of dark rearing depended on age. This bidirectional regulation was restricted to visual cortex and did not occur in frontal cortex. Bidirectional regulation was also specific to Munc13-3 and was not found for other Munc13 family members. Munc13 proteins serve a central priming function in synaptic vesicle exocytosis at glutamatergic and GABAergic synapses and this work contributes to the growing evidence indicating a role of Munc13 genes in synaptic plasticity.

Figure 1

Western blot analysis of regional distributions of Munc13 proteins in mouse brain. Samples from visual cortex (VC), frontal cortex (FC), and cerebellum (CB) were loaded and sequentially probed, stripped and reprobed with the Munc13 family member specific antibodies and with β-actin antibody to control for loading errors. Size standards in kDa are indicated on all western blots.

Figure 2

Analysis of Munc13-3 protein expression in mouse visual cortex and frontal cortex. Mice were reared in a normal environment (N) or complete darkness (D) until an age of 3.5 (N3.5, D3.5) or 9.5 weeks (N9.5, D9.5). Also shown are the same blots stripped and reprobed with a β-actin antibody to control for loading variations. Size standards in kDa are presented for all blots. Beneath are densitometric results for Munc13-3 protein levels in mouse visual and frontal cortex (corrected against β-actin) from three independent groups of mice. For each of the three groups of mice, values for each age/rearing condition were normalized against the highest value in each group. Means and standard errors from the three groups for each age/rearing condition are plotted for visual and frontal cortex.

Figure 3

Bidirectional regulation of expression was specific to Munc13-3. Western blot and densitometric results for Munc13-1 and Munc13-2 in mouse visual cortex are shown. Conventions as in figure 2.