Ultrastructural Evidence for a Role of Astrocytes and Glycogen-Derived Lactate in Learning-Dependent Synaptic Stabilization

Abstract

Long-term memory formation (LTM) is a process accompanied by energy-demanding structural changes at synapses and increased spine density. Concomitant increases in both spine volume and postsynaptic density (PSD) surface area have been suggested but never quantified in vivo by clear-cut experimental evidence. Using novel object recognition in mice as a learning task followed by 3D electron microscopy analysis, we demonstrate that LTM induced all aforementioned synaptic changes, together with an increase in the size of astrocytic glycogen granules, which are a source of lactate for neurons. The selective inhibition of glycogen metabolism in astrocytes impaired learning, affecting all the related synaptic changes. Intrahippocampal administration of l-lactate rescued the behavioral phenotype, along with spine density within 24 hours. Spine dynamics in hippocampal organotypic slices undergoing theta burst-induced long-term potentiation was similarly affected by inhibition of glycogen metabolism and rescued by l-lactate. These results suggest that learning primes astrocytic energy stores and signaling to sustain synaptic plasticity via l-lactate.

Keywords: 3D electron microscopy; glycogen metabolism; lactate; long-term memory; synaptic plasticity.

Figure 1

l-lactate rescues amnesia and hippocampal spine density impaired by DAB. (A) Schematic of the NOR experimental protocol. (B) Memory impairment was present in mice injected with 1000 and 2000 pmol DAB and administration of 100 nmol l-lactate in combination with 1000 pmol DAB, rescues amnesia in the NOR test. (C), (D) Images of 100 μm coronal section stained according to the Golgi-cox method. Scale bars, 300 μm and 100 μm. (E) Representative examples of Golgi-stained hippocampal neurons showing apical dendritic segments at high magnifications to highlight dendritic spines in mice control, injected with vehicle, 1000 pmol DAB, and 1000 pmol DAB +100 nmol l-lactate (scale bar: 10 μm). (F) Quantitation of spine number per 10 μm dendritic segment 24 hours from NOR training. (B), (F) ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 one-way analysis of variance, Tukey’s test (see Table S1).

Figure 2

DAB blockade of the LTP-dependent increase in spine formation and stabilization are rescued by l-lactate. (A) Individual spines on dendritic segments are imaged in transfected CA1 hippocampal pyramidal neurons. (B) Cch increased the percentage of eliminated spines (lost spines) at 24 hours after inducing of LTP in all experimental conditions. (C) Cch-induced LTP increase of the percentage of new spines after 24 hours was blocked with DAB and rescued by l-lactate. (D) Preexisting individual spines (left, white arrowhead) were either enlarging at 5 hours and stabilized over 4 days (yellow arrowhead) or nonenlarging at 5 hours (gray arrowhead). (E) Cch-induced selective spine stabilization was blocked by DAB and rescued by l-lactate. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. (B), (C) One-way analysis of variance, Kruskal-Wallis test. (E) Two-way analysis of variance. Scale bars: (A) left: 500 μm, right: 50 μm, bottom: 4 μm, (D) 2 μm (see Table S2).

Figure 3

l-lactate rescues DAB-inhibited formation of new spines but not spine and PSD size. (A) Example of three-dimensional reconstruction of dendritic spines heads (orange) in a hippocampal CA1 volume of 513.8 μm³. (B) DAB treatment significantly reduced the spine density 24 hours after NOR training and 100 nmol l-lactate coinjected with 1000 pmol DAB rescues the spine density. (C) 3D reconstruction of a spine head (orange) with its PSD (yellow); 3D-scale bar: 200 nm. Graph represent spine size measured with Blender, note the increase of the spine volume after learning (control vs. vehicle) and the decrease after DAB injection that is not rescued by l-lactate. (D) 3D reconstruction of a spine head (purple) with its PSD (yellow); 3D-scale bar: 200 nm. Graph represent PSD size measured with Blender, note the increase of the PSD size after learning (control vs. vehicle) and the decrease after DAB injection that is not rescued by l-lactate. (B), (C), (D) ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; one-way analysis of variance, Tukey’s test (see Table S1).

Figure 4

l-lactate does not rescue DAB-inhibited glycogen resynthesis. (A) TEM image of hippocampal excitatory synapses of mice treated with vehicle 24 hours after NOR training. In the astrocytic process contacting the presynaptic terminal, the glycogen granules are clearly visible, and the magnification shows the single granules inside the astrocyte. Scale bars: 100 nm and 50 nm. (B) Electron micrographs showing an increase of 221% in glycogen clusters’ size after learning in mice treated with vehicle versus control mice, and inhibitory effect of 1000 pmol DAB on glycogen granules’ resynthesis after learning that it is not rescued by l-lactate coadministration. Astrocytes processes are highlighted in blue and arrowheads indicated the glycogen granules’. Scale bar: 1 μm. (C) Corresponding violin plot of glycogen granules diameter. ^****P < 0.0001; one-way analysis of variance, Tukey’s test (see Table S1).

Figure 5

Schematics of the structural effects of LTP, its impairments by DAB and its rescue by l-lactate.