Functional relevance of the regulation of upper layer neuron numbers

The striking specificity of the phenotype in the AP2γ-/- cortex to occipital regions gave us the good possibility of studying the role of upper layer neurons in the function of the visual cortex. Indeed, exclusively in the occipital cortex, Cux1/2-positive upper layer neurons were reduced by about 40% and callosal projection neurons were also greatly affected after loss of AP2γ. This neurodevelopmental deficit of upper layer neurons in the occipital cortex of the adult AP2γ mutant mice had strong functional consequences in the visual performance of these mice. The visual acuity (cortical spatial resolution evaluated by VEPs) was abnormally low in AP2γ mutant mice as well as the ratio between contralateral and ipsilateral (C-I) that measures cortical binocularity by VEPs. There was also a trend for increased delay in the latency of visual responses. Indeed, visual acuity is a well-established measurement of the overall visual function in mammals (Fagiolini et al., 1994; Gianfranceschi et al., 1999). These

observations might be related with the reduction of callosal projections observed in these mice. Lmo4 is significantly downregulated in the AP2γ mutant cortex and it is known to be expressed in callosal neurons of layers II/III and in layer V (Arlotta et al., 2005; Bulchand et al., 2003), suggesting a role of this factor in the specification of callosal neurons.

Furthermore, Lmo4 plays a key role in mediating Ca2+ activation (Kashani et al., 2006) and may thereby impair important signalling pathways mediating functional neuronal maturation in the cerebral cortex. Importantly, Homer2, similarly downregulated in the AP2γ mutant, was shown to be expressed in pyramidal neurons of cortical layers II/III and V (Brakeman et al., 1997). Moreover, Emx1 was reported to be important for the proper formation of the corpus callosum. Consistently, Emx1 mutant mice lack most or all of their corpus callosum and the callosal commissure axons are stacked and failed to cross the midline into the opposite hemisphere (Hong et al., 2007; Qiu et al., 1996). Thus, Lmo4, Homer2 and Emx1 downregulation might contribute to the reduction of callosal neurons observed in this mutant. Taken together, the reduction in callosal connections in these mutant mice could play a role in their visual acuity capacities, further supporting the previous evidence for the importance of callosal projections in the development of visual acuity during the critical period in rats (Caleo et al., 2007).

Interestingly, visual acuity, binocularity and response latency develop progressively during maturation of the visual cortex. Indeed, the ratio between contralateral and ipsilateral is usually lower in juvenile mice as compared to adults (Frenkel and Bear, 2004; Sawtell et al., 2003). Moreover, response latency is higher in young versus fully mature animals ((Fagiolini et al., 1997). Thus, the lower C-I ratio in the adult AP2γ mutant mice might reflect an impairment in functional maturation of the cortex. The trend for an increased latency in the visual response in the AP2γ mutant is also consistent with an altered functional development. For example, similar functional deficits (low acuity, greater binocularity and increased latency) are observed when development is disturbed by rearing animals in the dark (Fagiolini et al., 1997). This data therefore prompt the hypothesis that the AP2γ mutant cortex might remain more plastic in the adult.

In agreement with this prediction, at the transcriptome level several genes upregulated by AP2γ at embryonic stages seem to be involved in plasticity. The pentraxin Ptx3, up-regulated in the E14 AP2γ mutant cortices at the transcriptome level, was already shown to have a protective role in seizure-induced neurodegeneration (Ravizza et al., 2001) and might contribute to synaptic plasticity (Xu et al., 2003). Indeed, some long pentraxins are expressed in the brain and some are involved in neuronal plasticity and degeneration. In addition, we

found the PDZ domain containing RING finger 3 (PDZRN3) being up-regulated in this mutant cortices. So far, there is no evidence for the function of PDZRN3 in the cortex, however, PDZRN3 was shown to function as a synapse-associated E3 ubiquitin ligase to regulate the postsynaptic development in other systems. Strikingly, this ubiquitin–proteasome pathway has been implicated in synaptic development and plasticity (Lu et al., 2007). Thus, the up-regulation of Ptx3 and PDZRN3 in the AP2γ mutant cortices at caudal levels may contribute to an increased plasticity in the adult visual cortex of these mutants, as suggested by the electrophysiology tests described above.

Strikingly, the neural cell recognition molecule Close Homolog of L1 (Chl1), up-regulated in the AP2γ mutant cortices at the transcriptome level, displays an interesting graded pattern of expression with highest levels caudally in the visual cortex, moderate levels in the somatosensory region, and low levels rostrally in the motor region (Liu et al., 2000). In the posterior cortical regions, Chl1 is higher expressed in the cortical plate and subventricular zone (Liu et al., 2000). Chl1 is required for neuronal positioning and dendritic growth of pyramidal neurons in the posterior regions of the developing mouse neocortex. Moreover, Chl1 is expressed in pyramidal neurons in a high-caudal to low-rostral gradient within the developing cortex (Demyanenko et al., 2004). Interestingly, deep layer pyramidal neurons of Chl1 knock-out mice are shifted to lower laminar positions in the visual and somatosensory cortex and developed misoriented. A downward shift in position of DiI-labelled callosal neurons in the middle layers (III/IV) was shown to occur in Chl1 mutants (Demyanenko et al., 2004). Thus, the restriction of Chl1 expression and the effects of its deletion in posterior neocortical areas suggest that Chl1 may regulate area-specific neuronal connectivity and have an important function in the visual cortex. Possibly, Chl1 is one candidate downstream target of AP2γ mediating its important neuronal functions in the visual cortex.