Impairment in Long-Term Memory Formation and Learning-Dependent Synaptic Plasticity in Mice Lacking Glycogen Synthase in the Brain

Abstract

Glycogen is the only carbohydrate reserve of the brain, but its overall contribution to brain functions remains unclear. Although it has traditionally been considered as an emergency energetic reservoir, increasing evidence points to a role of glycogen in the normal activity of the brain. To address this long-standing question, we generated a brain-specific Glycogen Synthase knockout (GYS1^Nestin-KO) mouse and studied the functional consequences of the lack of glycogen in the brain under alert behaving conditions. These animals showed a significant deficiency in the acquisition of an associative learning task and in the concomitant activity-dependent changes in hippocampal synaptic strength. Long-term potentiation (LTP) evoked in the hippocampal CA3-CA1 synapse was also decreased in behaving GYS1^Nestin-KO mice. These results unequivocally show a key role of brain glycogen in the proper acquisition of new motor and cognitive abilities and in the underlying changes in synaptic strength.

Keywords

brain glycogen hippocampus long-term potentiation mice operant conditioning

INTRODUCTION

The role of glycogen in the brain has been traditionally associated with the preservation of neuronal function during energetically challenging states such as hypoxia, hypoglycemia, or seizures. Nevertheless, glycogenolysis also occurs in euglycemia during an increase in neuronal activity, indicating that brain glycogen also has a role in supporting neuronal function in nonpathologic condition.^1–4 In this regard, glycogen concentration seems to be more conspicuous in areas of high synaptic density.^5,6

It has been proposed that the importance of brain glycogen lies in the rapidity of its breakdown compared with the uptake of extracellular glucose. During resting periods, glycogen pool would be replenished for further uses.⁷ This turnover could be crucial for learning and memory processes, where intense, energy-demanding increases in synaptic activity are required to induce the appropriate learning acquisition and storage mechanisms. Additionally, glycogen-derived pyruvate has been reported to be the preferred substrate for the de novo synthesis of glutamate.^8,9 Since glutamate-activated neurons are involved in learning consolidation,¹⁰ the relevance of glycogen in memory acquisition could go beyond mere energetic supply. Accordingly, several reports have described the importance of glycogenolysis for memory processing.^11,12

To unequivocally approach the study of the role of glycogen in the brain, here we generated a mouse model that lacks Glycogen Synthase specifically in the nervous system (GYS1^Nestin-KO). We analyzed the learning capacity of these animals and checked for differences in the electrophysiologic properties of the hippocampal CA3-CA1 synapse. We also characterized the levels of glycogen-related proteins in this model. Our results show the important contribution of brain glycogen to associative learning and to the concomitant changes in hippocampal synaptic strength, as well as to the experimental induction of long-term potentiation (LTP) in hippocampal synapses.

MATERIALS AND METHODS

Generation of GYS1 Conditional Mice

The GYS1 knockout-first C57BL/6N cells were obtained from the International Knockout Mouse Consortium. The cells were injected into C57BL/6J blastocysts, which were implanted in pseudo-pregnant C57BL/6J females for the generation of chimeric mice. One chimeric male positive for the disruption was mated with C57BL/6J females to test for germline transmission. Heterozygous F1 mice were crossed with mice expressing Flp Recombinase to eliminate the selection cassette and to generate the conditional allele.

Animal Studies

Five-month-old male mice were used in these experiments. Experimental protocols were performed following European Union Council (2003/65/EU) and Spanish (BOE 252/34367-91, 2005) guidelines for the use of laboratory animals in chronic studies and in accordance with ARRIVE guidelines. All experimental protocols were also approved by the Ethics Committee of the Pablo de Olavide University.

Surgery

Mice were anesthetized with 0.8% to 1.5% isoflurane, supplied from a calibrated Fluotec 5 (Fluotec-Ohmeda, Tewksbury, MA, USA) vaporizer, at a flow rate of 1 to 2 L/min oxygen (AstraZeneca, Madrid, Spain) and delivered by a mouse anesthesia mask (David Kopf Instruments, Tujunga, CA, USA). Animals were implanted with bipolar stimulating electrodes in the right Schaffer collateral-commissural pathway of the dorsal hippocampus (2 mm lateral and 1.5 mm posterior to Bregma; depth from the brain surface, 1.0 to 1.5 mm) and with a recording electrode in the ipsilateral CA1 area (1.2 mm lateral and 2.2 mm posterior to Bregma; depth from the brain surface, 1.0 to 1.5 mm).¹³ These electrodes were made of 50-μm, Teflon-coated tungsten wire (Advent Research Materials, Eynsham, UK). The recording electrode was implanted in the CA1 area using as a guide the field potential depth profile evoked by paired (40-ms interval) pulses presented to the ipsilateral Schaffer collateral pathway. The recording electrode was fixed at the site where a reliable monosynaptic (≤ 5 ms) field excitatory postsynaptic potential (fEPSP) was recorded. A bare silver wire was affixed to the skull as ground. Further details of these chronic preparations have been described elsewhere.¹⁴

Input/Output Curves, Paired-Pulse Facilitation and Long-Term Potentiation

For input/output curves, mice were stimulated at the hippocampal CA3-CA1 synapse with paired pulses (40 ms of interpulse interval) of increasing intensity (0.02 to 0.4 mA). Each pair of pulses was repeated ≥ 5 times with time intervals ≥ 30 seconds. For paired-pulse facilitation, we used pair of pulses of increasing intervals (10, 20, 40, 100, 200, and 500 ms) with intensity (in mA) fixed at ~40% of the amount necessary to reach asymptotic values. Long-term potentiation was evoked in behaving mice following procedures described previously.¹⁴ Field EPSP baseline values were collected 15 minutes before LTP induction using single 100 μs square, biphasic pulses. Pulse intensity was set at ~40% of the amount required to evoke a maximum fEPSP response (0.15 to 0.25 mA). For LTP induction, animals were presented with a high-frequency stimulation session. The high-frequency stimulation session consisted of five 200 Hz, 100 ms trains of pulses at a rate of 1/s repeated six times, at intervals of 1 minute. Thus, a total of 600 pulses were presented during each session of high-frequency stimulation. To avoid evoking large population spikes and the appearance of electroencephalographic seizures, the stimulus intensity during the high-frequency stimulation was set at the same as that used for generating baseline recordings. After the high-frequency stimulation session, the same single stimuli were presented every 20 seconds for 30 minutes and for the following 6 days.

Operant Conditioning

Training and testing were performed in Skinner box operant chambers. Before training, mice were handled daily for 7 days and food deprived to 80% of their free-feeding weight. Training took place for 20 minutes on successive days, in which mice learnt to press a lever to receive pellets from a food tray using a fixed-ratio (1:1) schedule. Field EPSPs evoked at the CA3-CA1 synapse were recorded during the training session. Stimuli were delivered at random (3 times/min), but for analysis only those presented when the animal was ready to press the lever were selected. A more complex conditioning task was performed for 11 days using a light/dark protocol, in which only during the lighted period was there a pellet reward. In addition, lever presses performed during the dark period restarted the dark protocol for additional time.^15,16

Biochemical Analyses

Animals were anesthetized and killed by decapitation. Brains were rapidly removed, frozen on liquid nitrogen and stored at −80°C until use. Total homogenates were analyzed for western blotting, and the determination of glycogen synthase activity and glycogen content as previously described.¹⁷

Immunohistochemistry

Five-month-old mice were anesthetized and perfused transcardiacally with phosphate-buffered saline (PBS) containing 4% (w/v) paraformaldehyde. Brains were removed, postfixed in the same solution overnight, cryoprotected with 30% (w/v) sucrose and sectioned at coronally planes (30 μM thick). For immunodetection of antigens, sections were washed in PBS and PBS-0.1% Triton X-100, blocked for 2 hours at room temperature with PBS containing 10% of normal goat serum and 0.2% of gelatin. Primary antibodies against GFAP (DAKO, Glostrup, Denmark), NeuN (Millipore, Billerica, MA, USA), calbindin and parvalbumin (Swant) were incubated overnight at 4°C with PBS-5% normal goat serum. Dye-labeled secondary antibodies and Hoechst 33342 were incubated for 2 hours at room temperature in 1 PBS-5% normal goat serum and mounted in Mowiol. Images were taken with a Nikon E1000 epifluorescence microscope (Nikon, Tokyo, Japan).

Data Analysis

Computed results were processed for statistical analysis using the IBM SPSS Statistics 18.0 (IBM, New York, NY, USA). Statistical significance of differences between groups was inferred by one-way ANOVA and ANOVA for repeated measures (data by groups), with a contrast analysis (Dunnett's post test) for a further study of significant differences. Statistical significance was set at P < 0.05.

RESULTS

Generation of a Brain-Specific GYS1 Knockout

In a first experimental step, we generated a conditional GYS1 allele that can be converted to a knockout (KO) allele by the action of a Cre Recombinase, which removes several exons of the gene (Figure 1A). To obtain a brain-specific GYS1 KO, we bred our conditional strain with Nestin-Cre transgenic mice to generate GYS1^Nestin-KO mice. The nestin promoter induces Cre recombinase expression in neuronal and glia cell precursors by embryonic day 11.¹⁸ Homozygous conditional, non-Cre-expressing littermates were used as controls (Figure 1B). Deletion of GYS1 had no effect on the morphology of the brain, as shown by histologic analyses of different brain regions with neuronal and astrocytic markers (Supplementary Material).

Figure 1.

Generation of a brain-specific Glycogen Synthase knockout (GYS1 KO). (A) Schematic representation of the alleles. The excision of the selection cassette from the knockout allele generates a conditional allele that expresses glycogen synthase normally. The subsequent excision of several exons from the conditional allele by a Cre Recombinase generates again a knockout allele. (B) Genotypes used in the study. Homozygous conditional, non-Cre-expressing littermates were used as controls of GYS1^Nestin ^KO. (C) Western blot of glycogen synthase in total homogenates of brain and skeletal muscle using an antibody that recognizes specifically the muscle (MGS) or the liver isoform (LGS). Mouse liver extract (L) was used as a positive control for LGS. Actin was used as a loading control. (D) Total glycogen synthase activity in the presence of its allosteric activator Glucose 6-phosphate (P = 2.80 × 10⁻⁷). (E) Glycogen levels (P = 0.007) in brain extracts of control and GYS1^Nestin-KO mice. Control (n = 4), KO (n = 4). *Statistically significant (P ≤ 0.05).

Analyses of brain extracts by western blot confirmed that GYS1^Nestin-KO mice are devoid of muscle Glycogen Synthase (MGS; Figure 1C). To rule out the possibility of the induction of the liver isoform of Glycogen Synthase to compensate for the absence of MGS, we analyzed liver isoform of Glycogen Synthase levels with a liver isoform-specific antibody, finding no expression. We also confirmed that our model is a brain-specific KO, as MGS levels in the skeletal muscle were equivalent in control and GYS1^Nestin-KO animals. As expected, Glycogen Synthase activity and glycogen levels were null in the brain extracts of GYS1^Nestin-KO mice (Figures 1D and 1E). These results prove that MGS is the only enzyme that synthesizes glycogen in the brain.

Analysis of the Levels of Proteins Involved in Glycogen Metabolism in the GYS1^Nestin-KO Mouse Brain

To study the impact of the absence of glycogen on the proteins involved in its metabolism, we analyzed changes in their expression by western blot (Figure 2) in total brain homogenates. Glycogen Phosphorylase isoforms expressed in the brain—brain and muscle isoforms—, total α/β and phosphorylated (inactive) β Glycogen Synthase Kinase 3 (GSK3 α/β and pGSK3 β, respectively) were unchanged. Contrary, glycogen debranching enzyme levels were decreased and AMP kinase (AMPK) increased in the GYS1^Nestin-KO brain (Figure 2A). In the case of AMPK, the levels of the phosphorylated (active) form (pAMPK) were unchanged. Glycogenin western blotting under normal conditions requires a previous treatment of the sample with amylase to digest the glycogen and thus free the protein so that it can be detected by the antibody.¹⁹ In the case of GYS1^Nestin-KO samples, however, glycogenin was detectable even without amylase treatment (Figure 2B). Equivalent results were obtained when analyzing changes in all proteins in isolated hippocampi (Supplementary Material).

Figure 2.

Changes in the expression of proteins involved in glycogen metabolism in the GYS1^Nestin-KO mouse brain. (A) MGS, brain and muscle isoforms of Glycogen Phosphorylase (BGPh and MGPh, respectively), glycogen debranching enzyme (GDE), AMP kinase (AMPK), pAMPK, GSK3 α/β, and pGSK3 β were analyzed in total brain homogenates. (B) Glycogenin levels were analyzed in untreated and amylase-treated total brain extracts. Actin was used as a loading control.

Differences in the Electrophysiologic Properties of the CA3-CA1 Synapse

To analyze the functional consequences of glycogen deficiency, we measured the response of hippocampal pyramidal CA1 neurons to paired pulses presented to the ipsilateral Schaffer collaterals in alert, behaving control and GYS1^Nestin-KO mice (Figure 3A). For this purpose, we first recorded the responses to paired pulses of increasing intensity. Both groups presented similar increases in the slope of fEPSPs evoked by the first pulse. These increases were best fitted by sigmoid curves (r ≥ 0.99; P < 0.0001), suggesting a normal functioning of the CA3-CA1 synapse in GYS1^Nestin-KO mice. Nevertheless, KO mice presented a larger facilitation to the second pulse than control mice (Figure 3B). Paired-pulse facilitation was further analyzed at a wide range of interpulse intervals. Interestingly, GYS1^Nestin-KO mice presented a significantly larger facilitation to paired pulses than control animals, especially at short interpulse intervals (F_(5,10) = 5.141; P < 0.001; Figure 3C).

Figure 3.

Electrophysiologic changes of hippocampal synapses in GYS1^Nestin-KO alert behaving mice. (A) Experimental design. Animals were chronically implanted with stimulating electrodes in the hippocampal Schaffer collaterals and with a recording electrode in the ipsilateral pyramidal CA1 area. An extra wire was attached to the bone as ground (DG, dentate gyrus; Sub., subiculum). (B) Input/output curves of field excitatory postsynaptic potentials (fEPSPs) evoked by paired pulses of increasing intensities in control (n = 11) and knockout (KO) (n = 11) mice. The best nonlinear adjustments to the collected data are illustrated. (C) Paired-pulse facilitation analyses (mean ± s.e.m. slopes of the second fEPSP expressed as a percentage of the first interpulse interval). Representative fEPSP paired traces (40 ms of interpulse interval) are shown on the right. Control (n = 11), KO (n = 11). (D) Time course of long-term potentiation (LTP) evoked in the CA3-CA1 synapse after a high-frequency stimulation (HFS) session (mean ± s.e.m. fEPSP slopes given as a percentage of values collected during baseline recordings (100%)). Control (n = 7), KO (n = 7). Representative examples of fEPSPs collected at the indicated times are plotted at the top. *Statistically significant (P < 0.05) differences between the two groups.

We next studied the LTP capabilities of the two animal groups. Long-term potentiation is widely considered to be the cellular basis of learning and memory.²⁰ After the high-frequency stimulation session, only the control group presented a significant LTP for the first recording day (F_(44,264) = 9.504; P < 0.001; Figure 3D). The GYS1^Nestin-KO mice did not present any evident sign of LTP (P ≥ 0.927) and even they showed a nonsignificant (P ≥ 0.879) depotentiation phenomenon during the subsequent recording days (Figure 3D). In addition, LTP values collected from controls were significantly (P < 0.05, see asterisks in Figure 3D) larger and longer lasting than those collected from GYS1^Nestin-KO mice. To discard a change in the levels of N-methyl-D-aspartate receptor (NMDAR) which could explain these differences, we analyzed the NR1 subunit of NMDA receptor (NMDAR1) by western blot and found no significant (P > 0.05) differences between the two groups. N-methyl-D-aspartate receptor forms a heterodimer of at least one NR1 and one NR2A-D subunit. Thus, by analyzing NMDAR1 subunit we addressed all possible combinations (see Supplementary Material). Thus, the deficit in LTP generation observed in GYS1^Nestin-KO mice could be ascribed to the unavailability of brain glycogen.

Impairment in the Acquisition of an Instrumental Conditioning Task and in the Associated Changes in Synaptic Strength

To further explore the role of brain glycogen, a highly demanding associative learning task (operant conditioning) was tested. Mice were trained in Skinner box modules to obtain a food pellet by pressing a lever located near the feeder (Figure 4A). In the first series of experiments, animals were rewarded with a food pellet every time the lever was pressed (fixed-ratio (1:1) schedule; Figure 4B, top diagram). Control animals reached the selected criterion—to press the lever a minimum of 20 times/session for two successive 20-minute sessions—earlier than GYS1^Nestin-KO mice (F_(6,72) = 4.629; P < 0.001; Figure 3C). The mean number of days needed (Figure 4D) and the percentage of mice reaching the selected criterion (Figure 4E) were also significantly different between groups. As already described, a proper functioning of hippocampal synaptic circuits is required for the acquisition of associative learning tasks.^14,15 Accordingly, the analysis of fEPSPs evoked at the hippocampal CA3-CA1 synapse during the learning process showed a significantly larger change in synaptic strength in controls when compared with values collected from GYS1^Nestin-KO mice (Figure 4F).

Figure 4.

Impaired performance of GYS1^Nestin-KO mice in an operant conditioning task. (A) Experimental set-up. Mice were trained in a Skinner box to press a lever to obtain a food pellet. (B) Two tasks of increasing difficulty were assayed. In task 1 (fixed-ratio (1:1)), mice received a food pellet each time they pressed the lever. In task 2 (Light/Dark), lever presses were rewarded only when a light bulb was switched on. Pressing the lever during the dark period punished the animal with an additional delay in the reappearance of the light period. (C) Lever presses in the first 7 days of training of task 1. Dotted line corresponds to criterion. (D) Mean days required to reach the criterion. (E) Percentage of mice reaching the criterion during the training. (F) Evolution of field excitatory postsynaptic potential (fEPSPs) evoked at the CA3-CA1 synapse during task 1 (mean ± s.e.m. fEPSP slopes given as a percentage of values collected before training (100%)). (G) Performance of control and GYS1^Nestin-KO mice during task 2. Control (CT) (n = 13), KO (n = 13). *Statistically significant (P < 0.01) differences between the two groups.

In a second series of experiments, animals that had reached the criterion, for the fixed-ratio (1:1) schedule, within 7 days were further trained in a more complex situation, in which they were rewarded only during the period in which a small light bulb was switched on (Figure 4B, bottom diagram). Pressing the lever during the dark period punished the animal with an additional delay in the reappearance of the lighted period. Again, control mice acquired this task faster than GYS1^Nestin-KO animals, reaching significant (F_(10,120) = 5.438; P < 0.01) differences by the tenth and eleventh training sessions (Figure 4G). In fact, GYS1^Nestin-KO did not reach the selected criterion (see dotted line in Figure 4G).

DISCUSSION

Here, we report for the first time on a mouse model lacking glycogen specifically in the brain. The use of a genetic model presents clear advantages over previous studies based on drug treatments, such as nonspecificity or uncontrolled side effects. Additionally, the tissue specificity of the gene deletion, as shown by the unaltered levels of Glycogen Synthase in the skeletal muscle, has allowed us to selectively study the involvement of brain glycogen in learning and memory processes.

We first studied how the lack of glycogen regulates the proteins related to its metabolism. Only glycogen debranching enzyme and AMPK showed changes in their protein levels, thus suggesting that their regulation is directly dependent on the presence of the glycogen molecule. Interestingly, the levels of phosphorylated AMPK remained unchanged, thereby pointing to a cellular compensation mechanism to maintain AMPK activity constant. Glycogenin was detectable in GYS1^Nestin-KO samples even without amylase treatment thereby further confirming the absence of glycogen. The difference in the mobility of glycogenin before and after amylase treatment may correspond to glycosyl units attached to the glycogenin protein by self-glycosylation.²¹

We next analyzed the functional consequences of the absence of brain glycogen. The histologic analysis of GYS1^Nestin-KO brain discarded developmental problems which could be on the base of the results obtained. Importantly, we performed electrophysiologic studies in alert, behaving animals, following procedures developed by some of us.^14,15,22 Our results show that paired-pulse facilitation, a well-characterized form of short-term plasticity related to short-term memory processes, is in fact enhanced in GYS1^Nestin-KO mice. Paired-pulse stimulation is frequently used as an indirect measurement of changes in the probability of neurotransmitter release at the presynaptic terminal.^23–25 It is assumed that any change in the response evoked by the second stimulus in relation to the first is indicative of a presynaptic action, while changes evoked simultaneously by both pulses are indicative of a postsynaptic effect.²⁶ Our findings thus reflect a disturbance in the release of neurotransmitters at the presynaptic terminal in GYS1^Nestin-KO mice. As recently shown in alert behaving mice,²⁷ this type of short-term modulation of synaptic strength can have a homeostatic role in the proper functioning of hippocampal circuits.²⁸ In this regard, the absence of appropriated postsynaptic LTP responses, presented by GYS1^Nestin-KO mice, is compensated in those animals by an increase in short-term presynaptic plasticity. As a whole, the present results are in accordance with previous reports pointing to a role for glycogen as a precursor of glutamate²⁹ and describing its importance in short-term memory processes.¹¹

However, LTP has been proposed as the cellular mechanism underlying the acquisition of new motor and cognitive abilities.^14,20,30 Remarkably, GYS1^Nestin-KO mice did not show any significant LTP after the presentation of the high-frequency stimulation session. As LTP acquisition implies high energy consumption, these results suggest that the availability of glycogen as a rapid energy source is crucial to evoke this long-lasting change in synaptic strength. The importance of brain glycogen could rely on the rapidity of its breakdown compared with uptake and phosphorylation of glucose. Indeed, the first step of glycolysis is glucose phosphorylation by Hexokinase, which is one of the slowest enzymes of the glycolytic pathway. Brain glycogen, in contrast, is metabolized by Glycogen Phosphorylase, a fast enzyme, generating glucose-6-phosphate. Thus, glycogen may provide supplemental fuel to support function when energy obtained from extracellular glucose is insufficient.⁷

Operant conditioning is a highly demanding associative learning task involving the participation of many cortical areas linked to spatial orientation, object recognition, coordinated motor responses, and cognitive processes relating ongoing behavior with its putative reward consequences. As already reported^15,16 and confirmed here, these analyses can be performed in mice when lever and feeder are adapted to their sizes. Results collected from the Skinner box show that the lack of brain glycogen results in a significant impairment in the learning process, as already suggested by the impairment in the experimental induction of LTP.

Overall, here we have shown that the lack of Glycogen Synthase, and thus of glycogen in the brain, produces a significant deficit in learning capacity and in the concomitant activity-dependent changes in synaptic strength. These observations unequivocally show the key role of brain glycogen in the proper and timed acquisition of relatively difficult learning tasks. This is the first time that the contribution of glycogen to learning has been unambiguously proven by means of genetic tools in alert behaving conditions. These findings may have relevant implications for the treatment of selective learning- and memory-related disorders.

DISCLOSURE/CONFLICT OF INTEREST

The authors declare no conflict of interest.

Footnotes

ACKNOWLEDGEMENTS

The authors thank María Sánchez-Enciso and Natàlia Plana for their technical assistance. Thanks also go to Stephen Forrow for advice and Tanya Yates for correcting the English manuscript.

Notes

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website ()

References

Sickmann

Walls

Schousboe

Bouman

Waagepetersen

. Functional significance of brain glycogen in sustaining glutamatergic neurotransmission. J Neurochem 2009; 109(Suppl 1): 80–86.

Dienel

Ball

Cruz

. A glycogen phosphorylase inhibitor selectively enhances local rates of glucose utilization in brain during sensory stimulation of conscious rats: implications for glycogen turnover. J Neurochem 2007; 102: 466–478.

Swanson

. Physiologic coupling of glial glycogen metabolism to neuronal activity in brain. Can J Physiol Pharmacol 1992; 70(Suppl): S138–S144.

Brown

Tekkok

Ransom

. Glycogen regulation and functional role in mouse white matter. J Physiol 2003; 549(Pt 2): 501–512.

Koizumi

Shiraishi

. Ultrastructural appearance of glycogen in the hypothalamus of the rabbit following chlorpromazine administration. Exp Brain Res 1970; 10: 276–282.

Phelps

. Barbiturate-induced glycogen accumulation in brain. An electron microscopic study. Brain Res 1972; 39: 225–234.

Shulman

Hyder

Rothman

. Cerebral energetics and the glycogen shunt: neurochemical basis of functional imaging. Proc Natl Acad Sci USA 2001; 98: 6417–6422.

Hertz

O'Dowd

Gibbs

. Reciprocal changes in forebrain contents of glycogen and of glutamate/glutamine during early memory consolidation in the day-old chick. Brain Res 2003; 994: 226–233.

Gibbs

Lloyd

Santa

Hertz

. Glycogen is a preferred glutamate precursor during learning in 1-day-old chick: biochemical and behavioral evidence. J Neurosci Res 2007; 85: 3326–3333.

10.

Schurr

Miller

Payne

Rigor

. An increase in lactate output by brain tissue serves to meet the energy needs of glutamate-activated neurons. J Neurosci 1999; 19: 34–39.

11.

Gibbs

Anderson

Hertz

. Inhibition of glycogenosis in astrocytes interrupts memory consolidation in young chickens. Glia 2006; 54: 214–222.

12.

Suzuki

Stern

Bozdagi

Huntley

Walker

Magistretti

. Astrocyte-neuron lactate transport is required for long-term memory formation. Cell 2011; 144: 810–823.

13.

Paxinos

Franklin

KBJ

. The Mouse Brain in Stereotaxic Coordinates. 2nd edn. Academic Press: San Diego, 2001.

14.

Gruart

Munoz

Delgado-Garcia

. Involvement of the CA3-CA1 synapse in the acquisition of associative learning in behaving mice. J Neurosci 2006; 26: 1077–1087.

15.

Madronal

Lopez-Aracil

Rangel

del Rio

Delgado-Garcia

Gruart

. Effects of enriched physical and social environments on motor performance, associative learning, and hippocampal neurogenesis in mice. PLoS ONE 2010; 5:e11130.

16.

Jurado-Parras

Gruart

Delgado-Garcia

. Observational learning in mice can be prevented by medial prefrontal cortex stimulation and enhanced by nucleus accumbens stimulation. Learn Mem 2012; 19: 99–106.

17.

Valles-Ortega

Duran

Garcia-Rocha

Bosch

Saez

Pujadas

. Neurodegeneration and functional impairments associated with glycogen synthase accumulation in a mouse model of Lafora disease. EMBO Mol Med 2011; 3: 667–681.

18.

Tronche

Kellendonk

Kretz

Gass

Anlag

Orban

. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat Genet 1999; 23: 99–103.

19.

Shearer

Graham

Battram

Robinson

Richter

Wilson

. Glycogenin activity and mRNA expression in response to volitional exhaustion in human skeletal muscle. J Appl Physiol 2005; 99: 957–962.

20.

Whitlock

Heynen

Shuler

Bear

. Learning induces long-term potentiation in the hippocampus. Science 2006; 313: 1093–1097.

21.

Cao

Steinrauf

Roach

. Mechanism of glycogenin self-glucosylation. Arch Biochem Biophys 1995; 319: 293–298.

22.

Gottlieb

Leal-Campanario

Campos-Esparza

Sanchez-Gomez

Alberdi

Arranz

. Neuroprotection by two polyphenols following excitotoxicity and experimental ischemia. Neurobiol Dis 2006; 23: 374–386.

23.

Zucker

Regehr

. Short-term synaptic plasticity. Annu Rev Physiol 2002; 64: 355–405.

24.

Fernandez de Sevilla

Cabezas

de Prada

Sanchez-Jimenez

Buno

. Selective muscarinic regulation of functional glutamatergic Schaffer collateral synapses in rat CA1 pyramidal neurons. J Physiol 2002; 545(Pt 1): 51–63.

25.

Lauri

Palmer

Segerstrale

Vesikansa

Taira

Collingridge

. Presynaptic mechanisms involved in the expression of STP and LTP at CA1 synapses in the hippocampus. Neuropharmacology 2007; 52: 1–11.

26.

Thomson

. Facilitation, augmentation and potentiation at central synapses. Trends Neurosci 2000; 23: 305–312.

27.

Madronal

Gruart

Delgado-Garcia

. Differing presynaptic contributions to LTP and associative learning in behaving mice. Front Behav Neurosci 2009; 3: 7.

28.

Turrigiano

Nelson

. Homeostatic plasticity in the developing nervous system. Nat Rev Neurosci 2004; 5: 97–107.

29.

Hertz

Gibbs

. What learning in day-old chickens can teach a neurochemist: focus on astrocyte metabolism. J Neurochem 2009; 109(Suppl 1): 10–16.

30.

Bliss

Collingridge

. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 1993; 361: 31–39.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

13.11 MB

Impairment in Long-Term Memory Formation and Learning-Dependent Synaptic Plasticity in Mice Lacking Glycogen Synthase in the Brain

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Generation of GYS1 Conditional Mice

Animal Studies

Surgery

Input/Output Curves, Paired-Pulse Facilitation and Long-Term Potentiation

Operant Conditioning

Biochemical Analyses

Immunohistochemistry

Data Analysis

RESULTS

Generation of a Brain-Specific GYS1 Knockout

Analysis of the Levels of Proteins Involved in Glycogen Metabolism in the GYS1Nestin-KO Mouse Brain

Differences in the Electrophysiologic Properties of the CA3-CA1 Synapse

Impairment in the Acquisition of an Instrumental Conditioning Task and in the Associated Changes in Synaptic Strength

DISCUSSION

DISCLOSURE/CONFLICT OF INTEREST

Footnotes

ACKNOWLEDGEMENTS

Notes

References

Supplementary Material

Analysis of the Levels of Proteins Involved in Glycogen Metabolism in the GYS1^Nestin-KO Mouse Brain