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. 2007 Jul;12(7):656-70.
doi: 10.1038/sj.mp.4001957. Epub 2007 Jan 30.

Hippocampus-specific deletion of BDNF in adult mice impairs spatial memory and extinction of aversive memories

S A Heldt  1 , L Stanek, J P Chhatwal, K J Ressler
Affiliations

Hippocampus-specific deletion of BDNF in adult mice impairs spatial memory and extinction of aversive memories

S A Heldt et al. Mol Psychiatry. 2007 Jul.

Abstract

Brain-derived neurotrophic factor (BDNF) is known to play a critical role in the synaptic plasticity underlying the acquisition and/or consolidation of certain forms of memory. Additionally, a role has been suggested for neurotrophin function within the hippocampus in protection from anxiety and depressive disorders. Understanding the function of this important gene in adult animals has been limited however, because standard knockouts are confounded by gene effects during development. There are no BDNF receptor-specific pharmacological agents, and infusions of neuropeptides or antibodies have other significant limitations. In these studies, we injected a lentivirus expressing Cre recombinase bilaterally into the dorsal hippocampus in adult mice floxed at the BDNF locus to facilitate the site-specific deletion of the BDNF gene in adult animals. Significant decreases in BDNF mRNA expression are demonstrated in the hippocampi of lenti-Cre-infected animals compared with control lenti-GFP-infected animals. Behaviorally, there were no significant effects of BDNF deletion on locomotion or baseline anxiety measured with startle. In contrast, hippocampal-specific BDNF deletions impair novel object recognition and spatial learning as demonstrated with the Morris water maze. Although there were no effects on the acquisition or expression fear, animals with BDNF deletions show significantly reduced extinction of conditioned fear as measured both with fear-potentiated startle and freezing. These data suggest that the cognitive deficits and impairment in extinction of aversive memory found in depression and anxiety disorders may be directly related to decreased hippocampal BDNF.

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Figures

Figure 1
Figure 1
In vitro and in vivo validation of LV-Cre virus. (a) HEK-293 cells transiently transfected with the green-red fluorescent cre-reporter plasmid, pLoxPGFP-DsRed and visualized 2 days later (−Cre), or infected with LV-Cre and visualized 1 day later (+ Cre, early (top), green filter; + Cre, early (bottom), red filter), or visualized 2 days later (+ Cre, late). (b) The floxed-stop lacZ reporter mice(129S-Gt(ROSA)26Sortm1Sor/J) were injected within dorsal hippocampus, and sections were processed for lacZ histochemistry with x-gal 14 days later to visualize Cre-dependent recombination in vivo. These demonstrate × 10 and × 20 magnification of LV-Cre-infected cells within CA1 and DG, illustrating the density and intact morphology of LV-Cre-infected hippocampal neurons. (c) BDNF (top) and Cre-recombinase (middle) mRNA expression visualized with in situ hybridization 2 weeks after LV-Cre infection into Bdnftm3Jae/J floxed mice. The bottom figure represents a pseudocolor overlay of the two in situ sections.
Figure 2
Figure 2
Reduction in BDNF mRNA in dorsal hippocampus with no changes in baseline activity or startle. (a) Qualitative figure showing BDNF in situ hybridization of dorsal hippocampus following a sham injection (top, −Cre) or following LV-Cre injection (bottom, + Cre). (b) Nissle-stained sections (from different experiment than (a) from hippocampi that have been infected with LV-GFP or LV-Cre. We found no discernable morphological changes with either viral infection compared to control. (c) Relative mRNA expression in dentate gyrus (DG), and CA1 and CA3 regions and the average of all regions (Avg) of dorsal hippocampus in LV-Cre (Cre)- or LV-GFP (GFP)-infected mice. (d) The estimated percentage of total area of each hippocampal region infected within the examined sections. (e) Baseline activity (%time moving) within the SR-LAB behavior box in mice that have been infected bilaterally with LV-Cre or LV-GFP. (f) Baseline acoustic startle reflex (startle amplitude) in mice that have been infected bilaterally with LV-Cre or LV-GFP (*P < 0.001).
Figure 3
Figure 3
Morris water maze and object recognition are impaired in hippocampal BDNF KO animals. (a) Acquisition of the location of the hidden platform, measured as the average latency to find the platform over several sessions of training, each separated by a day. LV-Cre-infected animals demonstrated significantly slower acquisition compared with LV-GFP-infected animals. (b) Following training, mice were tested on a probe trial in the absence of a platform. Number of crossings of the target location demonstrates that mice with LV-Cre injections made significantly fewer crossings over the target area than did LV-GFP controls. (c) Percent of time spent in the target, opposite or adjacent (average of both) quadrant during the probe test for each group of animals. (d) Percent of time spent exploring the new vs old object during the test day for novel object recognition. LV-GFP-infected animals spent significantly more time exploring the novel compared to the previously habituated object. The LV-Cre-infected animals did not differentiate between the two. (e) Scatter plot demonstrating a significant positive correlation t between animals performance on the MWM (number of target quadrant crossings) vs Novel Object Recognition (%time with novel object). LV-Cre-infected animals are represented with a diamond (N = 13), and LV-GFP animals with a square (N = 14). This correlation suggests that the manipulation of the dorsal hippocampus affected each spatial learning paradigm in a similar way (*P < 0.01; **P < 0.001).
Figure 4
Figure 4
Conditioned fear is not altered in mice with dorsal hippocampus BDNF deletions. (a) Acquisition of cue-conditioned fear, as measured with freezing after the onset of the auditory CS, during the conditioned fear acquisition session. There was no difference in acquisition of fear in animals receiving bilateral hippocampal injections of LV-Cre or LV-GFP. (b) Animals were tested within the same context in which training occurred for the presence of contextual fear as measured with freezing. There was no difference between the groups on level of contextual fear. (c) When animals were tested following acquisition in a different context, there was no difference in cue-conditioned fear as measured with FPS. (d) To examine explicitly the animals’ ability to discriminate the conditioned cue within a novel context, the level of acoustic startle reflex was measured during CS+ and CS− presentations of the acoustic startle stimulus. There is very tight overlap in the groups’ abilities to discriminate the fear-conditioned auditory cue (*P < 0.01).
Figure 5
Figure 5
Extinction of conditioned fear is impaired in mice with dorsal hippocampus BDNF deletions. (a) Percent FPS is graphed for the first post-fear training test (pre-extinction) vs the last test (post-extinction). Mice infected with LV-GFP demonstrate significant decreases in their level of conditioned fear as measured with %FPS compared with LV-Cre-infected mice. (b) Impaired extinction of fear, measured with %FPS, is stable across multiple testing sessions. (c) Percent freezing is graphed for the first post-fear training test (pre-extinction) vs the last test (post-extinction). The percent reduction in freezing from pre- to post-extinction is significantly greater for the LV-GFP group than the LV-Cre group. (d) Impaired extinction of fear measured with freezing demonstrated across testing sessions (*P < 0.05; **P < 0.005).
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