Sustained expression of brain-derived neurotrophic factor is required for maintenance of dendritic spines and normal behavior.

Brain-derived neurotrophic factor (BDNF) plays important roles in the development, maintenance, and plasticity of the mammalian forebrain. These functions include regulation of neuronal maturation and survival, axonal and dendritic arborization, synaptic efficacy, and modulation of complex behaviors including depression and spatial learning. Although analysis of mutant mice has helped establish essential developmental functions for BDNF, its requirement in the adult is less well documented. We have studied late-onset forebrain-specific BDNF knockout (CaMK-BDNF(KO)) mice, in which BDNF is lost primarily from the cortex and hippocampus in early adulthood, well after BDNF expression has begun in these structures. We found that although CaMK-BDNF(KO) mice grew at a normal rate and can survive more than a year, they had smaller brains than wild-type siblings. The CaMK-BDNF(KO) mice had generally normal behavior in tests for ataxia and anxiety, but displayed reduced spatial learning ability in the Morris water task and increased depression in the Porsolt swim test. These behavioral deficits were very similar to those we previously described in an early-onset forebrain-specific BDNF knockout. To identify an anatomical correlate of the abnormal behavior, we quantified dendritic spines in cortical neurons. The spine density of CaMK-BDNF(KO) mice was normal at P35, but by P84, there was a 30% reduction in spine density. The strong similarities we find between early- and late-onset BDNF knockouts suggest that BDNF signaling is required continuously in the CNS for the maintenance of some forebrain circuitry also affected by developmental BDNF depletion.

Learning deficits in forebrain-restricted brain-derived neurotrophic factor mutant mice.

Brain-derived neurotrophic factor (BDNF) participates in synaptic plasticity and the adaptive changes in the strength of communication between neurons thought to underlie aspects of behavioral adaptation. By selectively deleting BDNF from the forebrain of mice using the Cre site-specific DNA recombinase, we were able to study the requirements for BDNF in behaviors such as learning and anxiety. Early-onset forebrain-restricted BDNF mutant mice (Emx-BDNF(KO)) that develop in the absence of BDNF in the dorsal cortex, hippocampus, and parts of the ventral cortex and amygdala failed to learn the Morris Water Maze task, a hippocampal-dependent visuo-spatial learning task. Freezing during all phases of cued-contextual fear conditioning, a behavioral task designed to study hippocampal-dependent associative learning, was enhanced. These mice learned a brightness discrimination task well but were impaired in a more difficult pattern discrimination task. Emx-BDNF(KO) mice did not exhibit altered sensory processing and gating, as measured by the acoustic startle response or prepulse inhibition of the startle response. Although they were less active in an open-field arena, they did not show alterations in anxiety, as measured in the elevated-plus maze, black-white chamber or mirrored chamber tasks. Combined, these data indicate that although an absence of forebrain BDNF does not disrupt acoustic sensory processing or alter baseline anxiety, specific forms of learning are severely impaired.

Efficient bicistronic expression of cre in mammalian cells.

Cre recombinase-mediated DNA recombination is proving to be a powerful technique for the generation of mosaic mutant mice. To develop this technology further, we have altered the cre gene to enhance its expression in mammalian cells and have tested its efficiency of expression in a bicistronic message. Using a transient transfection assay, we found that the extension of a eukaryotic translation initiation consensus sequence, the insertion of two N-terminal amino acids, and the mutation of a cryptic splice acceptor site did not detectably alter Cre recombinase activity. The addition of either of two introns resulted in an approximately 2-fold increase in recombination frequency. We then tested the relative efficacy of Cre-mediated recombination in several bicistronic messages having the encephalomyocarditis virus internal ribosome entry site (IRES). Recombination frequencies were only reduced 2-fold relative to a comparable monocistronic cre gene. The latter results indicate that it will be possible to generate transgenic mouse strains having tissue-specific expression of the Cre recombinase through integration of an IRES-cre gene without disabling the targeted gene.

Involvement of class II beta-chain amino acid residues 85 and 86 in T-cell allorecognition.

Alloreactive T-cell clones were derived by limiting dilution following priming to allogeneic cells bearing HLA-DR1 alloantigens. Clonal specificities were determined by extensive testing on a panel of allogeneic lymphoblastoid cell lines and by blocking studies with monoclonal antibodies specific for HLA-DR, -DQ, and -DP class II molecules. Out of nine DR1-positive cell lines, three failed to stimulate a subset of the T-cell clones in conventional proliferation assays. Proliferation by all of the clones was blocked by anti-DR antibodies, not by anti-DQ or anti-DP, which was consistent with the conclusion that the HLA-DR molecule was recognized. This DR1-associated polymorphism has been identified as Dw20 by the Tenth International Histocompatibility Workshop. The molecular basis for this altered recognition of the DR1 molecule was determined by allele-specific oligonucleotide hybridization and by DNA sequencing studies. The first, second, and third hypervariable regions of all nine DR1-positive cell lines were identical. Valine and glycine were found at positions 85 and 86 of the DR1 beta 1 chain in DR1 molecules from six of the nine lymphoblastoid cell lines, whereas alanine and valine were found in the three variant (Dw20) DR1-positive cells. By analogy with class I structure, residues 85 and 86 would be located at the extreme C-terminal end of the beta-chain alpha helix. Together or separately, these amino acid differences may define a T-cell recognition element on the DR1 molecule serving to contact allospecific T-cell receptors. Alternatively, if allorecognition involves recognition of a self peptide complexed with an allogeneic MHC molecule, then it is possible that the differences T cells recognize on DR1 class II proteins arise from peptide-specific interactions with residues 85 and 86.