A role for autophagy in long-term spatial memory formation in male rodents

Abstract

A hallmark of long-term memory formation is the requirement for protein synthesis. Administration of protein synthesis inhibitors impairs long-term memory formation without influencing short-term memory. Rapamycin is a specific inhibitor of target of rapamycin complex 1 (TORC1) that has been shown to block protein synthesis and impair long-term memory. In addition to regulating protein synthesis, TORC1 also phosphorylates Unc-51-like autophagy activating kinase-1 (Ulk-1) to suppress autophagy. As autophagy can be activated by rapamycin (and rapamycin inhibits long-term memory), our aim was to test the hypothesis that autophagy inhibitors would enhance long-term memory. To examine if learning alters autophagosome number, we used male reporter mice carrying the GFP-LC3 transgene. Using these mice, we observed that training in the Morris water maze task increases the number of autophagosomes, a finding contrary to our expectations. For learning and memory studies, male Long Evans rats were used due to their relatively larger size (compared to mice), making it easier to perform intrahippocampal infusions in awake, moving animals. When the autophagy inhibitors 3-methyladenine (3-MA) or Spautin-1 were administered bilaterally into the hippocampii prior to training in the Morris water maze task, the drugs did not alter learning. In contrast, when memory was tested 24 hours later by a probe trial, significant impairments were observed. In addition, intrahippocampal infusion of an autophagy activator peptide (TAT-Beclin-1) improved long-term memory. These results indicate that autophagy is not necessary for learning, but is required for long-term memory formation.

Keywords: autophagy; hippocampus; long-term memory; water maze.

Figure 1. Spatial learning increases autophagy

A) PCR validation of the GFP-LC3 transgene in hemizygous mice. The wild-type gene is indicated by a 350 bp product, whereas the 250 bp band is amplified from the transgene. Hemizygous have both the 350 and 250 bp bands, corresponding to one wild-type allele and the transgene. B) Representative confocal images showing GFP immunoreactivity in the dendrites of CA1/CA2 pyramidal neurons from untrained and Morris water maze trained GFP-LC3 mice. Punctated GFP staining (arrows) appears to be increased as a result of behavioral training, suggesting increased autophagy. C) Confocal images of double-label immunohistochemistry showing colocalization (yellow) of GFP (green) and the autophagy protein Atg16L (red). Scale bars, 10μm. D) Summary results showing that the number of GFP-LC3 puncta was significantly increased as a result of spatial learning. Median is indicated by horizontal line. Error bars represent standard deviation. *, p<0.05.

Figure 2. Intrahippocampal administration of 3-MA or Spautin-1 inhibits autophagy

A) Representative images of western blots and B) summary data showing the immunoreactivity of class III PI3K, Beclin-1, LC3, and β-Actin in hippocampal extracts taken from an animal infused with 3-MA to one hippocampus while an equal volume of vehicle was infused to the contralateral hippocampus of the same animal. 3-MA significantly decreased LC3-II immunoreactivity. C) Representative western blots for class III PI3K, Beclin-1, LC3 and β-Actin in animals receiving intrahippocampal Spautin-1 to one hippocampus while simultaneously receiving vehicle to the contralateral hippocampus of the same animal. D) Summary data showing that Spautin-1 infusion significantly decreased LC3-II immunoreactivity as well as the levels of class III PI3K and Beclin-1. Median for each data set is indicated by horizontal line. Error bars represent standard deviation. Dotted line indicates vehicle value (100%). *, p<0.05.

Figure 3. Intrahippocampal infusion of 3-MA impairs long-term spatial memory

A) Rats were given bilateral infusions of either 3-MA (1.1. μl of 180 mM stock/side; n=9) or vehicle (n=11), then given 12 training trials in the Morris water maze task. Training data are presented as mean ± SEM. Memory was tested 24hr after training. Rats infused with 3-MA had impaired spatial memory as indicated by B) increased latency and C) fewer platform crossings. D) No differences between the groups were observed in swimming speed. Median for each data set is indicated by horizontal line. Error bars represent 90^th and 10^th percentile. *, p<0.05.

Figure 4. Intrahippocampal infusion of Spautin-1 impairs long-term spatial memory

A) Rats were given bilateral infusions of either Spautin-1 (1.1 μl of 450 μM stock/side; n=10) or vehicle (n=11). One hr later, rats were given 12 training trials in the abbreviated version of the Morris water maze task. Training data are presented as mean ± SEM. Memory was tested 24hr after training. Rats infused with Spautin-1 had impaired spatial memory as indicated by B) increased latency and C) fewer platform crossings. D) Swimming speed was not different between the Spautin-1- and vehicle-infused animals. Median for each data set is indicated by horizontal line. Error bars represent 90^th and 10^th percentile. *, p<0.05. E) Representative photomicrographs showing activated astrocytes (immunostained with GFAP) in the cortex immediately adjacent to the cannulation site when examined 2 weeks post-infusion. Distal to the cannulation site, ramified “resting” astrocytes were observed. Scale bar, 100μm. F) Representative photomicrographs of 2-week post-infusion hippocampi immunostained for NeuN (neurons), MAP2 (dendrites), and GFAP (astrocytes). No differences were observed between the vehicle- and Spautin-1-infused hippocampi. Scale bar, 1mm.

Figure 5. Post-training, intrahippocampal infusion of TAT-Beclin-1 improves long-term spatial memory

A) Rats were trained in the Morris water maze, then randomly assigned to receive either 6.0ng/side TAT-Beclin-1 peptide or an equimolar amount of TAT alone peptide. Training data are presented as mean ± SEM. Memory was tested 24hr after training. B) When tested for their memory, animals infused with TAT-Beclin-1 found the hidden platform significantly faster than those treated with TAT. Median for each data set is indicated by horizontal line. Error bars represent 90^th and 10^th percentile. *, p<0.05. C) Representative image of a cresyl violet stained tissue section indicating the terminus of the infusion (arrow heads). D) All infusion sites were found to be within the dorsal hippocampus (black dots).