The generation and characterisation of mice with conditional knock-out of the NMDA receptor subunit NR2B

The N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors. They play a critical role in neuronal pattern formation during development and in synaptic plasticity as molecular coincidence detectors. NMDA receptor is a tetrameric protein complex comprised of two obligatory NR1 subunits and two identical or different NR2 subunits, of which four types exist named NR2A-D. In rodents and other mammals, NR1 and NR2B are expressed in the entire central nervous system, already at embryonic stages, whereas NR2A expression starts and increases only postnatally to coexist with NR2B in the adult brain. Mice lacking the NR1 subunit or lacking the NR2B subunit die at birth, whereas mice lacking NR2A are viable. Both NR2A and NR2B containing NMDA receptors are implicated in synaptic plasticity, learning and memory formation but their distinctive functions are unknown. The NR2B subunit received a lot of attention because mice genetically altered to overexpress NR2B showed improved spatial reference memory and enhanced LTP. The lethality of the general NR2B knock-out gives rise to the necessity of a conditional knock-out, by which deleterious effects due to lack of NR2B during embryogenesis are prevented, and the physiological function of NR2B can be elucidated in the postnatal brain. For this purpose, a DNA construct for homologous recombination in embryonic stem (ES) cells was generated with NR2B allele exon 6 flanked by loxP sequences. This exon encodes the region preceding the first transmembrane domain of the NR2B subunit. As a selection marker, a neomycin resistance gene flanked by frt sites was introduced in intron 6. The selection marker was subsequently removed by flp recombinase from the targeted NR2B allele, and the ES cells were used for blastocyst injection to derive NR2B2lox mice. NR2B2lox mice were bred with TgCre4 mice, selectively expressing Cre recombinase in forebrain principal neurons, to generate mice heterozygous for the transgene TgCre4 and homozygous for the conditional NR2B allele (NR2B2lox/2lox). In these mice (NR2B(delta)Fb), postnatal forebrain principal neurons should lack NMDA receptors containing the NR2B subtype. Deletion of NR2B in NR2B(delta)Fb mice was revealed by electrophysiological measurements. In parallel, in this study, also lentiviral mediated gene delivery was used in vivo for Cre/loxP mediated DNA recombination. Recombinant lentivirus encoding Cre recombinase and the GFP protein under the control of the alpha-CaMKII promoter was delivered stereotactically to the hippocampal CA1 region of NR2B2lox/2lox mice at P20. Lack of NR2B was assessed by electrophysiological measurements of synaptic and whole-cell NMDA currents, using NR2B specific antagonists. Recordings from CA1 neurons revealed reduced NMDA currents, lack of sensitivity to ifenprodil, a selective blocker of NR2B containing NMDA receptors, and faster than wild type deactivation kinetics of NMDA mediated currents, indicating the effective loss of the NR2B-type NMDA receptors. Frequency and AMPA component of miniature EPSCs were unaltered whereas the NMDA component was reduced. Moreover an impairment of cellular LTP could be shown. Western blot analysis from hippocampal homogenates of NR2B(delta)Fb mice showed a 70% reduction of the NR2B subunit levels, and no significant change in the expression levels of NR2A, and of the AMPA receptor subunits GluR-A and GluR-B. Collectively, these studies describe a conditional mouse model for elucidating the particular physiological functions of the NR2B type of NMDA receptors in the adult forebrain