Increasing CRTC1 function in the dentate gyrus during memory formation or reactivation increases memory strength without compromising memory quality

Abstract

Memory stabilization following encoding (synaptic consolidation) or memory reactivation (reconsolidation) requires gene expression and protein synthesis (Dudai and Eisenberg, 2004; Tronson and Taylor, 2007; Nader and Einarsson, 2010; Alberini, 2011). Although consolidation and reconsolidation may be mediated by distinct molecular mechanisms (Lee et al., 2004), disrupting the function of the transcription factor CREB impairs both processes (Kida et al., 2002; Mamiya et al., 2009). Phosphorylation of CREB at Ser133 recruits CREB binding protein (CBP)/p300 coactivators to activate transcription (Chrivia et al., 1993; Parker et al., 1996). In addition to this well known mechanism, CREB regulated transcription coactivators (CRTCs), previously called transducers of regulated CREB (TORC) activity, stimulate CREB-mediated transcription, even in the absence of CREB phosphorylation. Recently, CRTC1 has been shown to undergo activity-dependent trafficking from synapses and dendrites to the nucleus in excitatory hippocampal neurons (Ch'ng et al., 2012). Despite being a powerful and specific coactivator of CREB, the role of CRTC in memory is virtually unexplored. To examine the effects of increasing CRTC levels, we used viral vectors to locally and acutely increase CRTC1 in the dorsal hippocampus dentate gyrus region of mice before training or memory reactivation in context fear conditioning. Overexpressing CRTC1 enhanced both memory consolidation and reconsolidation; CRTC1-mediated memory facilitation was context specific (did not generalize to nontrained context) and long lasting (observed after virally expressed CRTC1 dissipated). CREB overexpression produced strikingly similar effects. Therefore, increasing CRTC1 or CREB function is sufficient to enhance the strength of new, as well as established reactivated, memories without compromising memory quality.

Figure 1.

CRTC1 in the DG of the dorsal hippocampus. a, Similar to endogenous CRTC1 protein, plasmid-derived CRTC1 undergoes nuclear translocation following stimulation (KCl/FSK for 4 h) in primary hippocampal neurons. Scale bar, 20 μm. b, CRTC1 increased CRE-dependent transcription following stimulation in primary hippocampal neurons. (CRTC1, n = 4; GFP, n = 4). c, In the DG, CRTC1 is endogenously expressed exclusively in excitatory neurons (not interneurons or glia). Overlap of immunohistochemical staining for endogenous CRTC1 protein (red) and markers of different cell types: green, excitatory neurons (α-CaMKII); glia (GFAP); or interneurons (GAD67 or parvalbumin). Hoechst (blue) identifies DG granule cell layer. Merged images shows that endogenous CRTC1 protein is colocalized in cells positive for α-CaMKII (but not GFAP, GAD67, or parvalbumin). White arrows show lack of CRTC1 staining overlap in GAD67⁺ or parvalbumin⁺ cells. Scale bar, 50 μm. d, Context fear conditioning (CFC) increases c-Fos levels in mice microinjected with GFP vector and this effect is potentiated in mice microinjected with CRTC1 vector. CRTC1 overexpression had no effect on c-Fos levels homecage (HC) control mice (CFC–CRTC1, n = 4; CFC–GFP, n = 4; HC–CRTC1, n = 4, HC–GFP, n = 4). e, Input resistance, (f) resting potential, and (g) spike threshold (mV) did not differ between cells infected with CRTC1 vector or control cells whereas AHP (h, i) was decreased in cells infected with CRTC1 vector relative to control cells (CRTC1, n = 7; Control, n = 7). Means ± SEM.

Figure 2.

Microinjection of CRTC1 vector induces robust expression of CRTC1 in the DG. a, Vector microinjection induces robust localized transgene expression (GFP, green) in DG of dorsal hippocampus. Left, Coronal brain images (adapted from Paxinos and Franklin, 2001) depicting the AP extent of typical viral vector infection (−1.46 to −3.08 mm posterior to bregma). Right, Corresponding image showing transgene expression (GFP, green) following vector microinjection (assessed 4 d postmicroinjection; counterstained with DAPI, blue). Scale bar, 200 μm. b, HSV preferentially infects excitatory neurons (DG granule cells) (assessed 4 d postmicroinjection, top; counterstained with DAPI, blue). Scale bar, 50 μm. HSV-driven transgene expression dissipates by 10 d postmicroinjection (bottom). c, Microinjection of CRTC1 vector increases CRTC1 protein levels. Immunohistochemical staining for CRTC1 protein (red) in the DG 4 d following microinjection of GFP vector (top) or CRTC1 vector (bottom). Mice microinjected with CRTC1 vector show higher levels of CRTC1 protein levels than mice microinjected with GFP vector, in infected neurons (green). Scale bars: 200 μm (top); 50 μm (bottom).

Figure 3.

Locally and acutely increasing CRTC1 or CREB levels in DG during training enhance consolidation of contextual fear memory; this memory enhancement is context specific, not due to an effect on memory expression, and long lasting. a, Microinjection of CRTC1 or CREB vector in DG before weak training (1 × 0.3 mA shock) enhances contextual fear memory (CRTC1 vector, n = 29; CREB, n = 24; GFP, n = 27). This memory enhancement is specific for the training context (CXT-A), and does not generalize to a similar, nonshocked context (CXT-B), regardless of context testing order (CXT-A then CXT-B, left, or CXT-B then CXT-A, right). Mean ± SEM. b, Microinjection of CRTC1 or CREB vector in DG before strong training (3 × 0.5 mA shocks) enhances memory for contextual fear; this memory is specific to the training context (CXT-A) (CRTC1 vector, n = 10; CREB, n = 9; GFP, n = 8). c, Microinjection of CRTC1 or CREB vector after training does not facilitate memory expression (in either context) indicating that the enhancement of context memory by these vectors is not due to effects on memory expression/retrieval (CRTC1, n = 12; CREB, n = 11; GFP, n = 11). d, Memory enhancement produced by microinjection of CRTC1 or CREB vector is long lasting and maintains precision. Microinjection of CRTC1 or CREB vector before training (1 × 0.3 mA shock) enhances memory for contextual fear even when tested 30 d later (after transgene expression has dissipated) (CRTC1 vector, n = 12; CREB, n = 16; GFP, n = 9). This memory enhancement is context specific (only observed in CXT-A).

Figure 4.

Increasing CRTC1 or CREB levels in DG enhances reconsolidation of an established contextual fear memory. a, Microinjection of CRTC1 or CREB vector before reactivation of an established weak contextual fear memory enhances subsequent memory expression in a context-specific manner. Naive mice were trained with a weak protocol (1 × 0.3 mA shock), and 26 d later were microinjected with vector (CRTC1-R, n = 10; CREB-R, n = 10; GFP-R, n = 10). Three days following vector microinjection, all groups showed similar low levels of freezing when initially re-exposed to the training context (for 45 s) to reactivate the memory (left graph). In subsequent test session (24 h later), mice with GFP vector showed low levels of freezing (in both contexts). However, mice with CRTC1 or CREB vector showed enhanced memory, which was context specific (right graph). b, Memory reactivation is necessary for the enhancement of an established memory by CRTC1 or CREB vectors (reconsolidation). Mice were trained as above, similarly microinjected with vectors, but not re-exposed to the training context (no reactivation, NR) after vector microinjection (CRTC1-NR, n = 6; CREB-NR, n = 6; GFP-NR, n = 6). During the subsequent test, all groups showed equally low levels of freezing. Means ± SEM.