Identification of a functional connectome for long-term fear memory in mice.

Long-term memories are thought to depend upon the coordinated activation of a broad network of cortical and subcortical brain regions. However, the distributed nature of this representation has made it challenging to define the neural elements of the memory trace, and lesion and electrophysiological approaches provide only a narrow window into what is appreciated a much more global network. Here we used a global mapping approach to identify networks of brain regions activated following recall of long-term fear memories in mice. Analysis of Fos expression across 84 brain regions allowed us to identify regions that were co-active following memory recall. These analyses revealed that the functional organization of long-term fear memories depends on memory age and is altered in mutant mice that exhibit premature forgetting. Most importantly, these analyses indicate that long-term memory recall engages a network that has a distinct thalamic-hippocampal-cortical signature. This network is concurrently integrated and segregated and therefore has small-world properties, and contains hub-like regions in the prefrontal cortex and thalamus that may play privileged roles in memory expression.

Functional convergence of developmentally and adult-generated granule cells in dentate gyrus circuits supporting hippocampus-dependent memory.

In the hippocampus, the production of dentate granule cells (DGCs) persists into adulthood. As adult-generated neurons are thought to contribute to hippocampal memory processing, promoting adult neurogenesis therefore offers the potential for restoring mnemonic function in the aged or diseased brain. Within this regenerative context, one key issue is whether developmentally generated and adult-generated DGCs represent functionally equivalent or distinct neuronal populations. To address this, we labeled separate cohorts of developmentally generated and adult-generated DGCs and used immunohistochemical approaches to compare their integration into circuits supporting hippocampus-dependent memory in intact mice. First, in the water maze task, rates of integration of adult-generated DGCs were regulated by maturation, with maximal integration not occurring until DGCs were five or more weeks in age. Second, these rates of integration were equivalent for embryonically, postnatally, and adult-generated DGCs. Third, these findings generalized to another hippocampus-dependent task, contextual fear conditioning. Together, these experiments indicate that developmentally generated and adult-generated DGCs are integrated into hippocampal memory networks at similar rates, and suggest a functional equivalence between DGCs generated at different developmental stages.

Preferential incorporation of adult-generated granule cells into spatial memory networks in the dentate gyrus.

Throughout adulthood, new neurons are continuously added to the dentate gyrus, a hippocampal subregion that is important in spatial learning. Whether these adult-generated granule cells become functionally integrated into memory networks is not known. We used immunohistochemical approaches to visualize the recruitment of new neurons into circuits supporting water maze memory in intact mice. We show that as new granule cells mature, they are increasingly likely to be incorporated into circuits supporting spatial memory. By the time the cells are 4 or more weeks of age, they are more likely than existing granule cells to be recruited into circuits supporting spatial memory. This preferential recruitment supports the idea that new neurons make a unique contribution to memory processing in the dentate gyrus.

Development and validation of a sensitive entropy-based measure for the water maze.

In the water maze, mice are trained to navigate to an escape platform located below the water's surface, and spatial learning is most commonly evaluated in a probe test in which the platform is removed from the pool. While contemporary tracking software provides precise positional information of mice for the duration of the probe test, existing performance measures (e.g., percent quadrant time, platform crossings) fail to exploit fully the richness of this positional data. Using the concept of entropy (H), here we develop a new measure that considers both how focused the search is and the degree to which searching is centered on the former platform location. To evaluate how H performs compared to existing measures of water maze performance we compiled five separate databases, containing more than 1600 mouse probe tests. Random selection of individual trials from respective databases then allowed us to simulate experiments with varying sample and effect sizes. Using this Monte Carlo-based method, we found that H outperformed existing measures in its ability to detect group differences over a range of sample or effect sizes. Additionally, we validated the new measure using three models of experimentally induced hippocampal dysfunction: (1) complete hippocampal lesions, (2) genetic deletion of alphaCaMKII, a gene implicated in hippocampal behavioral and synaptic plasticity, and (3) a mouse model of Alzheimer's disease. Together, these data indicate that H offers greater sensitivity than existing measures, most likely because it exploits the richness of the precise positional information of the mouse throughout the probe test.

Imaging activation of adult-generated granule cells in spatial memory.

New neurons are continuously generated in the subgranular zone of the hippocampus throughout adulthood, and there is increasing interest as to whether these new neurons become functionally integrated into memory circuits. This protocol describes the immunohistochemical procedures to visualize the recruitment of new neurons into circuits supporting spatial memory in intact mice. To label adult-generated granule cells, mice are injected with the proliferation marker 5-bromo-2'-deoxyuridine (BrdU). At different delays after BrdU treatment, mice are trained to locate a hidden platform in the Morris water maze, and spatial memory can then be tested in a probe test with the platform removed from the pool. Ninety minutes after this probe test, mice are perfused and tissue is sectioned. Immunohistochemical procedures are used to quantify BrdU-labeled cells and expression of the immediate early gene, Fos. Because Fos expression is regulated by neuronal activity, the degree of overlap between BrdU-labeled and Fos-labeled neurons provides an indication of whether adult-generated granule neurons have been incorporated into spatial memory circuits.