4. Discussion
4.8 Embryonic HA-NRG1 overexpression induces hyperactivity, but no anxiety-like behavior
‘Global’ CRD-NRG1 overexpression in HA-Nrg1-tg mice lead to pronounced anxiety-like behavior and impaired PPI (Agarwal et al., 2014), as well as working memory deficits in the Y-maze test (Wehr et al., in preparation). To examine specific effects of
the temporal and spatial aspects of NRG1 overexpression on behavior, Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice were analyzed in the same set of behavior tests.
Unexpectedly, no signs of anxiety-like behavior were found in both conditional models. Instead, both lines showed hyperactivity, which is thought to correspond to psychomotor agitation in schizophrenic patients and is considered a schizophrenia-relevant behavior (Snyder, 1973; Yin et al., 2013a). Hyperactivity was found in the open field and the hole board test and was more pronounced in Stop-Nrg1*NEX-Cre mice. In the hole board test hyperactivity even seemed to prevent mice from exploring the new environment, as they showed tendencies to reduced exploration time and hole visits. Also in the tail suspension test Stop-Nrg1*NEX-Cre mice fought more against the aversive situation, in line with hyperactivity. Stop-Nrg1*NEX-Cre mice showed the strongest hyperactivity always at the beginning of a test, arguing for novelty-induced hyperactivity. In line with these findings, hypoactivity was observed in conditional CKII-Cre*Nrg1f/f (Agarwal et al., 2014) and Nestin-Cre*ErbB4null mutants (Golub et al., 2004). Hyperactivity was also described in conditional transgenic mice with modest Ig-NRG1 overexpression in the cortex (Yin et al., 2013a), interestingly, these mice also show no anxiety-like behavior. However, transgenic mice with Thy1.2-driven strong cortical and subcortical Ig-NRG1 overexpression show normal motor behavior with initial hypoactivity (Deakin et al., 2009). These contrasting findings in transgenic mice expressing the same NRG1 isoform. Indicate that differences in expression levels (and thereby ErbB4 stimulation) or cortical versus subcortical functions play an important role in NRG1-mediated regulation of motor behavior. NRG1 expression level differences might differentially regulate serotonine and/or dopamine signaling in the striatum and basal ganglia, including the Nucleus accumbens, thought to be involved in regulation hyperlocomotion (Taepavarapruk et al., 2000; Bishop and Walker, 2003; Brus et al., 2004; Fadda et al., 2005). Interestingly, these circuits might also be involved in the regulation of anxiety levels in these mice (Scott et al., 2006; Jiang et al., 2015).
No PPI impairments were observed in Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice, in contrast to HA-Nrg1-tg mice (Agarwal et al., 2014). In addition to unaltered PPI, both lines exhibited normal startle responses. Absence of PPI impairments could be related to lower cortical overexpression of HA-NRG1 in both lines compared to HA-Nrg1-tg mice, which is associated with a lower level of ErbB4 hyperphosphorylation. This idea is supported by the finding that administration of spironolactone ameliorates the PPI deficits in HA-Nrg1-tg mice. Spironolactone is a novel modulator of ErbB4 activity, which restores normal levels of ErbB4
phosphorylation in the brain of HA-Nrg1-tg mice (Wehr et al., in preparation). In addition, absence of HA-NRG1 overexpression from subcortical regions in conditional mouse lines, notably the striatal, could explain normal PPI as striatal projections have been considered important for the generation of PPI (Baldan Ramsey et al., 2011). PPI deficits appear not to be NRG1 isoform-specific as both Thy1.2 promoter-driven (Deakin et al., 2009) and conditional transgenic mice with Ig-Nrg1 overexpression exhibit impaired PPI (Yin et al., 2013). Finally, in line with a bell-shaped model of NRG1-mediated regulation of sensorimotor gating, PPI deficits also occur in heterozygous CRD-NRG1 mutants (Chen et al., 2008). Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice also showed no deficits in working memory, as measured in the Y-maze test. This is in contrasts to reduced performance of HA-Nrg1-tg mice in this test. Similar to PPI, administation of spironolactone to HA-Nrg1-tg mice ameliorates this deficit (Wehr et al., in preparation), suggesting that the level of NRG1/ErbB4 hyperstimulation is critical for the magnitude of working memory deficits.
Finally, deficits were observed in cued fear memory in Stop-Nrg1*NEX-Cre mice. A tendency to reduced contextual fear memory was also observed. Impaired contextual and cued fear conditioning was also found in CKII-Cre*Nrg1f/f mice (Agarwal et al., 2014), again supporting a bell-shaped model of NRG1 functions in hippocampal learning. However, considering the hyperactivity observed in Stop-Nrg1*NEX-Cre mice, deficits in fear conditioning could be independent from hippocampal learning deficits, but simply linked to hyperactivity-mediated reduction in freezing behavior.
In contrast to HA-Nrg1-tg mice (Wehr et al., in preparation), Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice showed normal pain sensitivity in the hotplate test, arguing for effects of Thy1.2 promoter-driven expression of HA-Nrg1 in the PNS.
Similar to Stop-Nrg1*NEX-Cre mice, Stop-Nrg1*CKII-Cre mice displayed a tendency for hyperactivity in the open field and hole board test, but performance in all other tests was unaltered. Thus, postnatal overexpression of HA-NRG1 exerts only minor effects on mouse behavior. However, as the Stop-Nrg1*CKII-Cre sample was small (n=7 Stop-Nrg1*CKII-Cre mice), this experiment can only be considered as a pilot study, which requires replication in a larger cohort.