Posttraining ablation of adult-generated neurons degrades previously acquired memories

Abstract

New neurons are continuously generated in the subgranular zone of the adult hippocampus and, once sufficiently mature, are thought to integrate into hippocampal memory circuits. However, whether they play an essential role in subsequent memory expression is not known. Previous studies have shown that suppression of adult neurogenesis often (but not always) impairs subsequent hippocampus-dependent learning (i.e., produces anterograde effects). A major challenge for these studies is that these new neurons represent only a small subpopulation of all dentate granule cells, and so there is large potential for either partial or complete compensation by granule cells generated earlier on during development. A potentially more powerful approach to investigate this question would be to ablate adult-generated neurons after they have already become part of a memory trace (i.e., retrograde effects). Here we developed a diphtheria toxin-based strategy in mice that allowed us to selectively ablate a population of predominantly mature, adult-generated neurons either before or after learning, without affecting ongoing neurogenesis. Removal of these neurons before learning did not prevent the formation of new contextual fear or water maze memories. In contrast, removal of an equivalent population after learning degraded existing contextual fear and water maze memories, without affecting nonhippocampal memory. Ablation of these adult-generated neurons even 1 month after learning produced equivalent memory degradation in the water maze. These retrograde effects suggest that adult-generated neurons form a critical and enduring component of hippocampal memory traces.

Figure 1.

DT-based ablation. a, Schematic of the tag and ablate strategy for ablating mature, adult-generated neurons. In adult nestin-Cre^ERT2+/iDTR⁺ mice (2xTg), TAM administration leads to permanent expression of DTRs in neural progenitor cells and their progeny. Subsequent administration of DT ablates this tagged population of adult-generated neurons only. b, c, In vitro assay demonstrating insensitivity of wild-type mouse cell lines to DT. Whereas mouse-derived 3T3 cells were insensitive to increasing concentrations of DT (n = 2) (b), DT dose-dependently reduced the number and viability of monkey-derived 2-2 cells (n = 3) (c).

Figure 2.

Cre^ERT2 expression is restricted to progenitor cells and limited to adult neurogenic regions. a, In nestin-Cre^ERT2+ mice, Cre^ERT2 protein expression (green) was found in nestin⁺ and DCX⁺ cells (red) but not mature neurons (NeuN; red) in the DG (scale bar, 10 μm). b–g, In these mice, Cre^ERT2 protein expression was limited to the subgranular zone of the DG and the subventricular zone of the lateral ventricle (LV) (millimeters relative to bregma; scale bars, 250 μm). Mo, Molecular layer; GCL, granule cell layer; CPu, caudate–putamen; Pir, piriform cortex; Thl, thalamus; OB, olfactory bulb; Gr, granular layer; Gl, glomerular layer; CA, cornu ammonis.

Figure 3.

Tagging new neurons. a, TAM-induced recombination occurs only in nestin-Cre^ERT2+ mice (scale bar, 50 μm). b, Recombination occurred throughout the anteroposterior extent of the DG (millimeter relative to bregma; scale bars, 150 μm). c, Schematic showing markers associated with different developmental stages of adult hippocampal neurogenesis. d, Seven weeks after the completion of TAM treatment, most LacZ⁺ cells (green) costained for mature neuronal markers (NeuN, calbindin; red), with far less staining for progenitor cell or immature neuronal markers (nestin, DCX, calretinin; red). LacZ⁺ cells additionally expressed activity-dependent gene zif268 (red) after behavioral testing. e, Seven weeks after the completion of TAM treatment, Ki67 expression levels were similar in control (CTR; n = 11) and 2xTg (n = 7) mice, indicating that DTR expression has no effect on ongoing proliferative activity in the adult hippocampus (scale bar, 250 μm). Mo, Molecular layer; GCL, granule cell layer.

Figure 4.

Ablating tagged neurons. a, DT (n = 4) but not PBS (n = 6) efficiently ablated DTR-expressing cells in the DG (scale bar, 10 μm). b–d, Consistent with this, DT treatment reduced overall numbers of doublecortin⁺ (DCX; scale bar, 150 μm) (b), nestin⁺ (scale bar, 10 μm) (c), and calretinin⁺ (scale bar, 10 μm) (d) cells in the DG of 2xTg mice. Mo, Molecular layer; GCL, granule cell layer. *p < 0.05.

Figure 5.

DT-induced ablation produces minimal inflammation. a, GFAP (red) and ionized calcium binding adaptor molecule 1 (Iba1; green) expression in CTR (n = 5) and 2xTg (n = 4) mice 24 h after administration of DT. b, GFAP levels were similar for CTR and 2xTg mice in both the DG and CA1 regions. c, Iba1 expression was increased in 2xTg mice only in the DG and not in the CA1 region. Note that Iba1 expression was mainly limited to the subgranular zone and innermost layer of the DG, a pattern that matches the distribution of tagged (i.e., DTR⁺ or LacZ⁺) cells after TAM treatment. *p < 0.05.

Figure 6.

General health and behavior are not altered by DT-induced ablation. Seven weeks after the completion of TAM treatment, CTR (n = 6) and 2xTg (n = 7) mice were treated with DT. a, Body weights were not different after the completion of TAM or DT treatments. b–j, The behavior of TAM- and DT-treated CTR and 2xTg mice was characterized in a battery of tests. We observed no effect on time spent immobile in the forced swim test (b); total exploration in the open field (c); time spent in the outer, middle, and innermost regions of the open field (d); latency to find platform in the visual discrimination water maze (WM) (e); swim speed during training in the visual discrimination water maze (f); paw slips in the beam walk test (g); latency to fall in the bar hanging test (h); latency to detect adhesive tape in the sticky dot test (i); or latency to remove adhesive tape in the sticky dot test (j).

Figure 7.

Posttraining ablation of adult-generated neurons degrades contextual fear memory. a, Mice were treated with TAM and trained in context A. After DT-induced ablation of adult-generated neurons, contextual memory was assessed in contexts A–C. b, c, During training, CTR (n = 12) and 2xTg (n = 11) mice exhibited similar response to shock (b) and freezing (c) levels before and after shock delivery. d, After DT treatment, CTR mice froze more in the trained context (A) versus a similar context (B). In contrast, 2xTg mice froze equally in both. e, DT-induced ablation abolished context discrimination. f, g, Freezing in a dissimilar context (C) (f) and tone fear (g) were similar in CTR and 2xTg mice. h, Mice were treated with TAM and trained in a conditioned taste aversion task. During training, saccharin was paired with 0.15 m LiCl. After DT-induced ablation of adult-generated neurons, preference for saccharin versus water was evaluated. CTR (n = 12) and 2xTg (n = 11) mice exhibited equivalent preference for saccharin. Importantly, this preference was lower compared with mice (n = 11) for which saccharin was paired with saline (rather than LiCl) during training. *p < 0.05.

Figure 8.

Pretraining ablation of adult-generated neurons does not prevent the formation of a new contextual fear memory. a, Mice were trained in context A after DT-induced ablation of adult-generated neurons. Contextual memory was assessed in contexts A–C 1 week later. b, c, During training, CTR (n = 13) and 2xTg (n = 14) mice exhibited similar response to shock (b) and freezing (c) levels before and after shock delivery. d, e, After DT treatment, both CTR and 2xTg mice discriminated between contexts A and B (d), and the degree of discrimination did not differ (e). f, g, Freezing in a dissimilar context (f) and tone fear (g) were equivalent in CTR and 2xTg mice. *p < 0.05.

Figure 9.

Posttraining (but not pretraining) ablation of adult-generated neurons impairs spatial memory expression. a, Mice were treated with TAM and trained in the water maze. After DT-induced ablation of adult-generated neurons, spatial memory was assessed in a probe test. b, During training, latency to find platform declined equivalently in CTR (n = 12) and 2xTg (n = 12) mice. c, After DT-induced ablation, 2xTg mice searched less selectively compared with CTR mice, spending less time in the target zone (T). d, Additional groups of TAM-treated mice were trained in the water maze. However, mice were treated with PBS (rather than DT) during the week preceding memory testing. e, During training, latency to find platform declined equivalently in CTR (n = 11) and 2xTg (n = 14) mice. f, In the probe test, both CTR and 2xTg mice searched selectively, spending more time in the target zone compared with other (O) nontarget zones in the pool. g, Mice were trained in the hidden version of the water maze after the completion of TAM treatment. During the week before training, mice were treated with DT. h, During training, latency to find the platform declined equivalently in CTR (n = 14) and 2xTg (n = 11) mice. i, In the probe test, both CTR and 2xTg mice searched selectively at the target zone. j, Mice were treated with TAM and trained in the water maze. One month later, mice were treated with DT, and spatial memory was assessed in a probe test. k, During training, latency to find platform declined equivalently in CTR (n = 14) and 2xTg (n = 12) mice. l, After DT-induced ablation at the remote time point, 2xTg searched less selectively compared with CTR mice, spending less time in the target zone. *p < 0.05.

Figure 10.

Posttraining ablation of adult-generated neurons impairs visual discrimination memory. a, During training, a submerged platform was located beneath one of two visual cues (e.g., horizontal stripes). After training, the hippocampus was lesioned, and discrimination between the reinforced and nonreinforced cues was evaluated in a probe test. b, Before surgery, latency to locate the platform declined at similar rates in lesion (n = 10) and sham (n = 9) mice. c, After surgery, whereas sham mice searched selectively at the reinforced cue, lesion mice did not search at either cue. d, Representative images of brains from sham and lesion mice. e, CTR (n = 12) and 2xTg (n = 11) mice were treated with TAM and trained in the visual discrimination task. After DT-induced ablation of adult-generated neurons, visual discrimination memory was assessed in a probe test. f, Across training days, latency to locate the platform declined at similar rates in CTR and 2xTg mice. g, In the probe test, whereas CTR mice searched selectively at the reinforced cue, 2xTg mice spent equivalent amounts of time close to the reinforced and nonreinforced cues. h, Heat maps reflect preferential searching close to previously reinforced (left peak) versus nonreinforced (right peak) cue in CTR but not 2xTg mice. Note that the peak at top of pool corresponds to the release point at the start of the probe test. *p < 0.05.