Effect of context exposure after fear learning on memory generalization in mice.

BACKGROUND: The conditions under which memory generalization occurs are not well understood. Although it is believed that fear memory generalization is gradually established after learning, it is not clear whether experiences soon after learning affect generalization. RESULTS: Using a contextual fear conditioning paradigm in mice, we found that fear memory generalization occurred when mice were exposed to a familiar, unconditioned context soon after fear learning. CONCLUSIONS: Our results suggest that the familiarity of contexts and the timing of their exposure influences memory generalization, which increases our understanding of the mechanisms of generalization.

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.

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.

Involvement of the anterior cingulate cortex in the expression of remote spatial memory.

Although the hippocampus plays a crucial role in the formation of spatial memories, as these memories mature they may become additionally (or even exclusively) dependent on extrahippocampal structures. However, the identity of these extrahippocampal structures that support remote spatial memory is currently not known. Using a Morris water-maze task, we show that the anterior cingulate cortex (ACC) plays a key role in the expression of remote spatial memories in mice. To first evaluate whether the ACC is activated after the recall of spatial memory, we examined the expression of the immediate early gene, c-fos, in the ACC. Fos expression was elevated after expression of a remote (1 month old), but not recent (1 d old), water-maze memory, suggesting that ACC plays an increasingly important role as a function of time. Consistent with the gene expression data, targeted pharmacological inactivation of the ACC with the sodium channel blocker lidocaine blocked expression of remote, but spared recent, spatial memory. In contrast, inactivation of the dorsal hippocampus disrupted expression of spatial memory, regardless of its age. We further showed that inactivation of the ACC blocked expression of remote spatial memory in two different mouse strains, after training with either a hidden or visible platform in a constant location, and using the AMPA receptor antagonist CNQX. Together, our data provide evidence that circuits supporting spatial memory are reorganized in a time-dependent manner, and establish that activity in neurons intrinsic to the ACC is critical for processing remote spatial memories.

BrdU assay for neurogenesis in rodents.

Neurogenesis within the adult central nervous system is demonstrated using an exogenous cell tracer, 5'-bromo-2'-deoxyuridine (BrdU), in combination with endogenous neuronal markers. Specific primary antibodies raised against these markers are widely available and their visualization is possible with the use of fluorescently tagged secondary antibodies. BrdU is a thymidine analog that incorporates into dividing cells during DNA synthesis. Once incorporated into the new DNA, BrdU will remain in place and be passed down to daughter cells following division. Typically, BrdU is injected intraperitoneally. Different survival times required by the desired experimental time-line will yield data on specific phases of neurogenesis: proliferation, differentiation and maturation. One of the drawbacks of using BrdU is the dependence on a stressful injection procedure and uncertain penetration of the targeted cells with a uniform concentration of the compound. Thus, for experiments requiring measurements of cell proliferation, Ki67 can be used as an acceptable alternative. The protocol takes 3-5 d, allowing for sectioning and staining.

Electrophysiological correlates of neural plasticity compensating for ischemia-induced damage in the hippocampus.

Injury to the brain often results in loss of synapses or cell death in the damaged area. Subsequent to the injury, the areas that are not directly affected often exhibit enhanced neuronal plasticity. Although there are many reports of morphological changes resulting from such plasticity, their functional consequences are poorly understood. In this study we examined electrophysiological changes associated with ischemia-induced neurogenesis in the hippocampus, a brain region that is particularly vulnerable but also exceptionally plastic. Transient global ischemia was induced in Sprague-Dawley rats by occlusion of both carotid arteries and a reduction in blood pressure for 12 min. The procedure resulted in delayed cell death in the CA1 field of the hippocampus while the dentate gyrus (DG) was spared. To assess neurogenesis and synaptic changes in parallel we used both hemispheres from each animal. One side was used for immunohistochemistry and the other for in vitro electrophysiological experiments in brain slices. Synaptic field responses and synaptic plasticity (LTP) in perforant path within the DG were reduced by 50% at 10 days after the ischemic injury but recovered at 35 days. Synaptic responses in non-neurogenic CA1 were abolished in parallel with cell death and did not recover. Gamma irradiation applied focally to the head selectively prevented neurogenesis and the synaptic recovery in the DG. These experiments reveal electrophysiological changes associated with reactive neural plasticity in the hippocampus.