C 57-DBA strain differences in presynaptic function 1 Strain differences in presynaptic function : proteomics , ultrastructure and physiology of hippocampal synapses in DBA / 2 J and C 57 Bl / 6 J mice

The inbred strains C57BL/6J (C57) and DBA/2J (DBA) display striking differences in a number of behavioral tasks depending on hippocampal function, such as contextual memory. Historically, this has been explained through differences in post-synaptic protein expression underlying synaptic transmission and plasticity. We measured the synaptic hippocampal protein content (iTRAQ and mass spectrometry), CA1 synapse ultrastructural morphology and synaptic functioning in adult C57 and DBA mice. DBA mice showed a prominent decrease in the Ras-GAP calciumsensing protein RASAL1. Furthermore, expression of several presynaptic markers involved in exocytosis, such as syntaxin (Stx1b), Ras-related proteins (Rab3a/c), and rabphilin (Rph3a), was reduced. Ultrastructural analysis of CA1 hippocampal synapses showed a significantly lower number of synaptic vesicles and presynaptic cluster size in DBA mice, without changes in postsynaptic density or active zone. In line with this compromised presynaptic morphological and molecular phenotype in DBA mice, we found significantly lower paired-pulse facilitation and enhanced short-term depression of glutamatergic synapses, indicating a difference in transmitter release and/or refilling mechanisms. Taken together, our data suggest that in addition to strain-specific post-synaptic differences, the change in dynamic properties of presynaptic transmitter release may underlie compromised synaptic processing related to cognitive functioning in DBA mice. Inbred and gene-specific mutant mouse strains have provided a basis for exploration of the impact of genetic variation on structural, physiological and molecular factors that modulate learning and memory. A number of strains display prominent differences in behavioral tasks depending on hippocampal function (1). In particular, DBA/2J (DBA) is outperformed by the C57Bl/6J (C57) strain in tasks for aversive memory formation and maintenance (2). Various deficits in tasks for spatial memory, such as Barnes and Morris water maze (3–5), and non-spatial memory, such as cued and contextual fear conditioning (5, 6), have been reported for DBA mice. These memory impairments may be linked to a reduced capacity http://www.jbc. org/cgi/doi/10.1074/jbc.M114.628776 The latest version is at JBC Papers in Press. Published on April 24, 2015 as Manuscript M114.628776 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. at U N IV D E SH E R B R O O K E on M ay 2, 2015 hp://w w w . jb.org/ D ow nladed from C57-DBA strain differences in presynaptic function 2 for the maintenance of theta-burst LTP in the CA1 area of the hippocampus (Schaffer collateral pathway) in DBA compared with C57 (5, 7). Theta-burst activity is assumed important in novel context exploration (8), an important factor in the formation of spatial and fear conditioning memories. Induction of LTP appeared unaffected unless interburst intervals were extremely compressed to three seconds (8, 9). In search of the principal molecular mechanisms underlying the physiological and behavioral phenotype of DBA mice, studies have mainly focused on postsynaptic proteins. For instance, changes in the level and activity of signaling proteins such as protein kinase C (PKC), the protein kinase A-pathway and mitogen activated protein kinase specifically affect behavioral and physiological performance (10, 11). Differences in the level and activity of some of the above proteins have also been shown to affect memory formation in DBA mice (11–13). For example, treatment with oxiracetam increased membrane-bound PKC, which was associated with improved memory performance of DBA mice in the Morris water maze (14). However, the diverse physiological responses to the temporal pattern of stimulation may be insufficiently explained by postsynaptic mechanisms only. A reduced capacity for pairedpulse facilitation (PPF) has been observed (5), which suggests the presence of changes in presynaptic dynamics and short-term plasticity that may further contribute to the physiological and behavioral phenotype in DBA mice. Since a comprehensive view of the synaptic composition, morphology and short-term plasticity in these strains is lacking, we performed an integrative analysis of the synaptic differences between DBA and C57 in adult (10–13 weeks) mice. We observed decreased expression levels of presynaptic markers involved in exocytosis, together with a lower number of synaptic vesicles and presynaptic cluster size in DBA mice, and no changes in postsynaptic density or active zone. Moreover, DBA mice displayed decreased pairedpulse facilitation and enhanced short-term depression of glutamatergic synapses. These results suggest an alteration in transmitter release or refilling mechanisms that, in addition to known postsynaptic mechanisms, may contribute to learning and memory differences in these strains. EXPERIMENTAL PROCEDURES Animals. Male C57Bl6/J and DBA/2J were obtained from Charles River (Lyon), and were individually housed with cage enrichment and water and food ad libitum on a 12/12 h rhythm lights on/off with lights on at 7 AM. All experiments were approved by the Animal Users Care Committee of the VU University and complied with the European Council Directive