Impaired OD plasticity in adult Bsn-/- mice

The protein Bassoon is not only present in ribbon synapses but also in conventional synapses in the brain and important for assembling the active zone of the presynaptic membrane.

Hence, Bassoon mutant mice showed a reduction in normal synaptic transmission, which might be due to the inactivation of a part of glutamatergic synapses (Altrock et al., 2003).

Therefore Bassoon plays a role in the regulation of neurotransmitter release from a subset of glutamatergic synapses (Altrock et al., 2003). Since inhibition plays a major role in OD plasticity (Hensch et al., 1998) and Bassoon mutant mice showed an excitation-inhibition-imbalance and impaired striatal plasticity (Ghiglieri et al., 2009), we wanted to investigate whether mutant mice also display impaired neuronal plasticity in the visual cortex.

131 Intrinsic optical imaging of V1 revealed a significant shift of OD due to decreased deprived-eye responses after four days of MD in juvenile Bsn-/- mice similar to their juvenile Bsn+/+ littermates. Interestingly, OD plasticity was absent in adult Bsn-/- animals in contrast to their wild-type littermates, which displayed a significant shift of OD due to increased open-eye responses after seven days of MD.

Juvenile (P20) as well as adult (P60) Bassoon mutant mice suffer from epileptiform seizures, whereas adult mice display up to twice as much and more severe seizures as juvenile mice (Altrock et al., 2003; Ghiglieri et al., 2009). Based on these findings the physiological and morphological impairments due to the seizures might be more severe in adult than in juvenile mutant animals, which would fit to our data.

Bassoon mutant mice showed decreased LTP in corticostriatal synapses (Ghiglieri et al., 2009), which is due to the presence of epileptiform seizures. LTP plays a major role in neuronal plasticity by increasing cortical responses of the open eye after MD (Espinosa and Stryker, 2012). This might also be an explanation for the absent OD plasticity in the visual cortex in adult mutant mice. The impairment of LTP might be the result of altered NMDAR subunit composition in mutants (Ghiglieri et al., 2009). In fact, activation, accurate assembly, and localization of NMDARs in the cytoplasmic surface below the postsynaptic membrane of glutamatergic synapses termed postsynaptic density (PSD), are essential for LTP (Picconi et al., 2004) whereas the PSD is altered in brain areas with occurring seizures (Wyneken et al., 2001; Wyneken et al., 2003; Finardi et al., 2006). Bassoon mutant mice showed altered NMDAR organization in the striatal PSD, NR2A and NR2B levels were modified and revealed an increase in the NR2A/NR2B ratio (Ghiglieri et al., 2009), this might also be the case in V1.

The regulation of the NR2A/NR2B ratio plays a crucial role in the control of cortical synaptic plasticity (Yashiro and Philpot, 2008). In the visual cortex of mice a reduction of the NMDAR subunit NR2A enhances the activity-dependent strengthening of synapses during MD (Cho et al., 2009). Chow and her colleagues (2009) hypothesized that a reduction in the NR2A/NR2B ratio during MD is permissive for the compensatory potentiation of open-eye inputs. In contrast, Bassoon mutant mice showed an increase in NR2A/NR2B ratio, which one could conclude that this increase leads to hindered or even absent potentiation of open-eye responses, like we observed in adult Bassoon mutant mice.

132 Monocular deprivation in adult mice increases presynaptic release of glutamate (Yashiro et al., 2005). Hippocampal glutamatergic synapses in Bsn-/- mice are partly inactive (up to 50 % more inactive synapses than in wild-type animals), which leads to a fewer number of neurons that are able to release neurotransmitters (Altrock et al., 2003). It is not clear whether Bassoon mutant mice show this increase in glutamate release during MD and if so this increase of the remaining active synapses might not be sufficient to induce OD plasticity.

Interestingly, immunohistochemistry revealed that juvenile as well as adult Bassoon mutant mice displayed a higher number of parvalbumin positive fast-spiking interneurons in the visual cortex, which was previously only reported for the striatum (Ghiglieri et al., 2009).

Additionally, we found that this number increased during age. As previously reported Bassoon mutant mice display an excitation-inhibition-imbalance partly due to a reduction of excitatory drive onto GABAergic interneurons, which may lead to lower excitation of interneurons and therefore to decreased GABA release (Altrock et al., 2003). This finding is most likely the reason for epileptiform seizures and seizures can cause extensive neuronal loss and severe brain damage (Olney et al., 1983; Sloviter, 1983, 1996). To prevent this kind of damage, the brain might adapt by increasing inhibition via an increased number of PV interneurons possibly by an augmented activation of silent/inactive interneurons, which did not yet express parvalbumin. Enhanced inhibition could be the explanation for the absent adult OD plasticity in Bassoon mutant mice. The conclusion that GABAergic inhibition limits adult plasticity in the visual cortex was drawn after pharmacological reduction of intracortical inhibition (obtained through infusion of an inhibitor of GABA synthesis or a GABAA antagonist directly into the visual cortex) reactivated OD plasticity in response to MD in adult rats (Harauzov et al., 2010).

Noteworthy, a recent study showed that Bassoon mutant mice displayed a highly elevated level of BDNF measured by ELISA (Heyden et al., 2011). Epileptic seizures are most likely the reason for increased BDNF levels and promoted neurogenesis (Parent et al., 1997; Lee et al., 2002; Altar et al., 2003; Yokoi et al., 2007). Previous studies revealed that BDNF

133 overexpression in transgenic mice induced premature start as well as termination of the critical period in the visual cortex (Hanover et al., 1999; Huang et al., 1999). BDNF is involved in regulating efficacy and number of inhibitory synapses, hence the amount of inhibition that pyramidal cells receive (Huang et al., 1999). If not only the critical period but also the period of adult plasticity would be terminated precociously, this might be an explanation for the phenotype of absent adult plasticity in adult but not in juvenile mutant mice. Unfortunately we couldn´t find this elevated level of BDNF with immunohistochemical staining. This might be because of different reasons: i) ELISA is a much more sensitive method to measure even small amounts of proteins than immunohistochemical staining, ii) Heyden and her colleagues (2011) measured the whole cerebral cortex whereas we only stained the visual cortex and there might not be that highly elevated level of BDNF present, or iii) the amount of BDNF antibody was too low to stain the whole amount of protein and therefore didn´t reveal any differences between Bsn+/+ and Bsn-/- mice.

The stability of synapses requires complex interactions with an extracellular matrix structure; the perineuronal nets (PNNs). PNNs form a lattice-like configuration around the synapses on the proximal and somata dendrites of certain neurons (Hockfield et al., 1990;

Brückner et al., 1993), and therefore influence synaptic integrity and stability (Frischknecht and Gundelfinger, 2012). Chondroitin sulfate proteoglycans, e.g. neurocan and brevican, are major components of the PNNs (Yamaguchi, 2000). Numerous studies revealed that chondroitin sulfate proteoglycans play a role in plasticity (Pizzorusso et al., 2002; Dityatev and Schachner, 2003; Pizzorusso et al., 2006; Tropea et al., 2009; Frischknecht and Gundelfinger, 2012). After degradation of chondroitin sulfate proteoglycans with chondroitinase-ABC in adult rats, OD plasticity was reactivated and MD caused an OD shift toward the nondeprived eye (Pizzorusso et al., 2002). Thus, the mature extracellular matrix is inhibitory for experience-dependent plasticity. Status epilepticus, a state in which the brain is in persistent seizure, leads to an increased expression of neurocan and brevican (Kurazono et al., 2001; Matsui et al., 2002; Yuan et al., 2002; Galtrey and Fawcett, 2007;

McRae et al., 2012; McRae and Porter, 2012) and might therefore prevent neuronal plasticity. This would fit with our observation of the absent OD plasticity in adult Bassoon mutant mice. From our data it is not clear if the absent adult OD plasticity is due to the mutation in the Bassoon gene per se, or whether the frequent epileptiform seizures in the mutant mice affect the OD plasticity.

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