Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing methods
Aim. Evidence-backed execution summary for Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing methods from Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing.
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Surgeries
reagent used in the protocol.
- Use
- Rats were anesthetized with isoflurane and placed in a stereotaxic frame. In studies of the effects on memory of intrahippocampal drug infusions, two 8-mm guide cannulae (Plastics One) were chronically implanted bilaterally above the central portion of the ventral hippocampus (coordinates: 5.5 mm posterior and ±...
Bioprobe measurements of extracellular hippocampal glucose and lactate during behavioral testing
reagent used in the protocol.
- Use
- Either a glucose- or a lactate-sensitive biosensor was inserted into the dorsal hippocampus via a guide cannula (Pinnacle Technology Inc., Lawrence, KS). The biosensor projected 3 mm beyond the end of the implanted cannula. The last 1 mm of the probe was coated with lactate oxidase or glucose oxidase to metabolize t...
Intrahippocampal Injections to enhance or impair memory
reagent used in the protocol.
- Use
- All injections (0.5 µl, 0.9% saline vehicle, pH = 7.2) were made bilaterally into the ventral hippocampus with a CMA/100 microinjection pump at a flow rate of 0.25 µl/min 5 min prior to behavioral testing. Ten rats were injected with lactate (0, 50, 100, or 150 nmol of lactate in 0.5 µl of...
Histology for glycogen localization and for cannula placements
reagent used in the protocol.
- Use
- On completion of testing, rats received an overdose of sodium pentobarbital followed by intracardial perfusion with ice-cold 4% paraformaldehyde in 0.1 M PBS. Brains were removed and postfixed overnight in 4% paraformaldehyde. They were then transferred to a 20% glycerol solution until they lost buoyancy (∼48...
Histology for glycogen localization and for cannula placements
reagent used in the protocol.
- Use
- To show that 4-CIN did not block monocarboxylate transport into mitochondria, activity of the mitochondria was monitored using a stain for succinate dehydrogenase (SDH) activity,. SDH activity has previously been demonstrated to decrease in the presence of 3-bromopyruvate and with sensory deprivation,, supportin...
Memory testing
Spatial working memory was assessed using spontaneous alternation tasks,,. Spontaneous alternation was chosen to assess memory because it requires no food reward and thus no food restriction; therefore the natural levels of glucose or lactate would be at baseline at the start of testing. The task was also chosen...
- Use
- Spatial working memory was assessed using spontaneous alternation tasks,,. Spontaneous alternation was chosen to assess memory because it requires no food reward and thus no food restriction; therefore the natural levels of glucose or lactate would be at baseline at the start of testing. The task was also chosen...
Memory testing
All other pharmacological experiments examined impairments of memory with drugs that block lactate delivery from astrocytes to neurons, together with possible reversals of these impairments with co-administration of glucose or lactate. No delay between arm entries was imposed during these experiments or in the exper...
- Use
- All other pharmacological experiments examined impairments of memory with drugs that block lactate delivery from astrocytes to neurons, together with possible reversals of these impairments with co-administration of glucose or lactate. No delay between arm entries was imposed during these experiments or in the exper...
Memory testing
The pharmacological manipulations were performed as within-subjects testing of multiple doses. Each testing session occurred in a new room with new extramaze visual cues to encourage sufficient exploratory behavior. Additionally, an interval of at least 48 hrs was imposed between testing sessions to allow sufficient...
- Use
- The pharmacological manipulations were performed as within-subjects testing of multiple doses. Each testing session occurred in a new room with new extramaze visual cues to encourage sufficient exploratory behavior. Additionally, an interval of at least 48 hrs was imposed between testing sessions to allow sufficient...
Bioprobe measurements of extracellular hippocampal glucose and lactate during behavioral testing
Either a glucose- or a lactate-sensitive biosensor was inserted into the dorsal hippocampus via a guide cannula (Pinnacle Technology Inc., Lawrence, KS). The biosensor projected 3 mm beyond the end of the implanted cannula. The last 1 mm of the probe was coated with lactate oxidase or glucose oxidase to metabolize t...
- Use
- Either a glucose- or a lactate-sensitive biosensor was inserted into the dorsal hippocampus via a guide cannula (Pinnacle Technology Inc., Lawrence, KS). The biosensor projected 3 mm beyond the end of the implanted cannula. The last 1 mm of the probe was coated with lactate oxidase or glucose oxidase to metabolize t...
Intrahippocampal Injections to enhance or impair memory
All injections (0.5 µl, 0.9% saline vehicle, pH = 7.2) were made bilaterally into the ventral hippocampus with a CMA/100 microinjection pump at a flow rate of 0.25 µl/min 5 min prior to behavioral testing. Ten rats were injected with lactate (0, 50, 100, or 150 nmol of lactate in 0.5 µl of...
- Use
- All injections (0.5 µl, 0.9% saline vehicle, pH = 7.2) were made bilaterally into the ventral hippocampus with a CMA/100 microinjection pump at a flow rate of 0.25 µl/min 5 min prior to behavioral testing. Ten rats were injected with lactate (0, 50, 100, or 150 nmol of lactate in 0.5 µl of...
Histology for glycogen localization and for cannula placements
On completion of testing, rats received an overdose of sodium pentobarbital followed by intracardial perfusion with ice-cold 4% paraformaldehyde in 0.1 M PBS. Brains were removed and postfixed overnight in 4% paraformaldehyde. They were then transferred to a 20% glycerol solution until they lost buoyancy (∼48...
- Use
- On completion of testing, rats received an overdose of sodium pentobarbital followed by intracardial perfusion with ice-cold 4% paraformaldehyde in 0.1 M PBS. Brains were removed and postfixed overnight in 4% paraformaldehyde. They were then transferred to a 20% glycerol solution until they lost buoyancy (∼48...
Histology for glycogen localization and for cannula placements
Forty-µm sections were also taken from the same brains around the areas of cannula implantations to confirm placements using cresyl violet. Photomicrographs were taken using Image Pro Express (v. 5.1.0.12, Media Cybernetics, Inc., Bethesda, MD). No data had to be excluded from rats with either extensive tissue...
- Use
- Forty-µm sections were also taken from the same brains around the areas of cannula implantations to confirm placements using cresyl violet. Photomicrographs were taken using Image Pro Express (v. 5.1.0.12, Media Cybernetics, Inc., Bethesda, MD). No data had to be excluded from rats with either extensive tissue...
Lactate and glucose levels increase and decrease, respectively, during behavioral testing
Recordings from bioprobes before, during and after memory testing revealed substantial increases in extracellular hippocampus levels of lactate and decreases in extracellular levels of glucose beginning with the placement of the rat on the maze ( ). Extracellular lactate concentrations significantly increased in the...
- Use
- Recordings from bioprobes before, during and after memory testing revealed substantial increases in extracellular hippocampus levels of lactate and decreases in extracellular levels of glucose beginning with the placement of the rat on the maze ( ). Extracellular lactate concentrations significantly increased in the...
Bioprobe measurements of extracellular hippocampal glucose and lactate during behavioral testing
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Either a glucose- or a lactate-sensitive biosensor was inserted into the dorsal hippocampus via a guide cannula (Pinnacle Technology Inc., Lawrence, KS). The biosensor projected 3 mm beyond the end of the implanted cannula. The last 1 mm of the probe was coated with lactate oxidase or glucose oxidase to metabolize t...
Statistical Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All statistical analyses were done using SPSS v. 18.0 (SPSS, Chicago, IL). For each experiment, repeated measures ANOVAs were used to analyze differences. Fisher's least significant difference post hoc tests were conducted when the results from the ANOVAs were significant.
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Methods
Male, Sprague-Dawley rats (Harlan Laboratories; 3 months old at the time of surgery) were housed individually with free access to food and water and maintained on a 12∶12 hr light/dark cycle with lights on at 7:00 am. All procedures described in this paper were approved by the University of Illinois Urbana-Champaign Institutional Animal Care and Use Committee in accordance with guidelines outlined in Guide for Care and Use of Laboratory Animals and accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.
Surgeries
Rats were anesthetized with isoflurane and placed in a stereotaxic frame. In studies of the effects on memory of intrahippocampal drug infusions, two 8-mm guide cannulae (Plastics One) were chronically implanted bilaterally above the central portion of the ventral hippocampus (coordinates: 5.5 mm posterior and ±4.8 mm lateral from bregma and 4.2 mm ventral from skull) to accommodate infusion cannulae near the time of behavioral testing. Due to the shape and size of the housing for the wireless potentiostat used for measurements of changes in extracellular glucose and lactate levels during behavioral testing, the guide cannula for bioprobes needed to be placed close to the midline of the skull. Therefore the guide cannulae for bioprobes were implanted above the dorsal hippocampus instead of the ventral hippocampus in either the left or right hemisphere (coordinates: 3.8 mm posteri...
Memory testing
Spatial working memory was assessed using spontaneous alternation tasks,,. Spontaneous alternation was chosen to assess memory because it requires no food reward and thus no food restriction; therefore the natural levels of glucose or lactate would be at baseline at the start of testing. The task was also chosen because it measures spatial working memory, which is sensitive to hippocampal manipulations. In the current experiments, animals were placed on a four-arm, plus-shaped maze (arms: 45 cm long, 14 cm wide, 7.5 cm tall; center area: 14 × 14 cm) constructed of opaque, black Plexiglas, as described previously,, or in a four-arm, plus-shaped maze with slightly higher sides (45 cm long, 14 cm wide, and 15 cm tall; center area: 14 × 14 cm) that were made of clear Plexiglas. The maze with higher arms was used to contain the rats better on the maze in experiments in which a d...
Bioprobe measurements of extracellular hippocampal glucose and lactate during behavioral testing
Either a glucose- or a lactate-sensitive biosensor was inserted into the dorsal hippocampus via a guide cannula (Pinnacle Technology Inc., Lawrence, KS). The biosensor projected 3 mm beyond the end of the implanted cannula. The last 1 mm of the probe was coated with lactate oxidase or glucose oxidase to metabolize the respective analyte, generating a current measured by the probe. The biosensor was connected to a potentiostat inside the head cap, which sent readings of the current generated by lactate or glucose in extracellular fluid to a computer by telemetry, with data recording and storage in 1-sec bins accomplished with Pinnacle Technology Laboratory v. 1.6.7 software. Glucose biosensors have previously been shown to have a range of 0-10 mM with in vitro sensitivity of 1.6±0.4 nA/mM (mean ± SEM) and lactate biosensors have a range of 0-8 mM with an in vitro...
Intrahippocampal Injections to enhance or impair memory
All injections (0.5 µl, 0.9% saline vehicle, pH = 7.2) were made bilaterally into the ventral hippocampus with a CMA/100 microinjection pump at a flow rate of 0.25 µl/min 5 min prior to behavioral testing. Ten rats were injected with lactate (0, 50, 100, or 150 nmol of lactate in 0.5 µl of 0.9% saline, pH = 7.2), 12 rats were injected with 1,4-dideoxy-1,4-imino-D-aribinitol (DAB; 0, 0.5, 5, 50 pmol, 50 pmol+25 nmol glucose, or 50 pmol+50 nmol lactate in 0.5 µl of 0.9% saline, pH = 7.2), and 8 rats were injected with α-cyano-4-hydroxycinnamate (4-CIN; 3, 10, 30 pmol, 30 pmol+25 nmol glucose, or 30 pmol+50 nmol lactate in 0.5 µl of 1% DMSO and 0.9% saline, pH = 7.2) were infused into the hippocampus 5 min prior to behavioral testing. DAB, an inhibitor of glycogen phosphorylase, was used to block glycogenolysis...
Histology for glycogen localization and for cannula placements
On completion of testing, rats received an overdose of sodium pentobarbital followed by intracardial perfusion with ice-cold 4% paraformaldehyde in 0.1 M PBS. Brains were removed and postfixed overnight in 4% paraformaldehyde. They were then transferred to a 20% glycerol solution until they lost buoyancy (∼48 hrs). They were then sectioned on a cryostat (Leica CM1850, Leica Microsystems Inc., Germany) with 20 µm slices of prefrontal cortex, striatum, dorsal hippocampus, and ventral hippocampus saved for glycogen localization analysis using a periodic acid-Schiff's reagent method (PAS) previously demonstrated to highlight glycogen. Prior to PAS staining, immunofluorescence methods were used to stain neurons with neuronal nuclear antigen (NeuN) and astrocytes with glial fibrillary acidic protein (GFAP) so that all sections were stained with PAS and consecutive sections were...
Histology for glycogen localization and for cannula placements
To show that 4-CIN did not block monocarboxylate transport into mitochondria, activity of the mitochondria was monitored using a stain for succinate dehydrogenase (SDH) activity,. SDH activity has previously been demonstrated to decrease in the presence of 3-bromopyruvate and with sensory deprivation,, supporting a relationship between SDH activity and mitochondrial function. Prior to sacrifice, the rats used in the 4-CIN dose response experiment received 30 pmol 4-CIN unilaterally in the ventral hippocampus and 1% DMSO in saline in the other hemisphere, providing within-subjects comparisons. The hemispheres receiving 4-CIN were counterbalanced across rats. Five min after the injection animals were tested on spontaneous alternation for 20 minutes to parallel the other behavioral experiments. The rats then received an overdose of sodium pentobarbital and were perfused transcardiall...
Statistical Analysis
All statistical analyses were done using SPSS v. 18.0 (SPSS, Chicago, IL). For each experiment, repeated measures ANOVAs were used to analyze differences. Fisher's least significant difference post hoc tests were conducted when the results from the ANOVAs were significant.
Measurement outputs
What raw and processed outputs should exist?
Spatial working memory was assessed using spontaneous alternation tasks,,. Spontaneous alternation was chosen to assess memory because it requires no food reward and thus no...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The pharmacological manipulations were performed as within-subjects testing of multiple doses. Each testing session occurred in a new room with new extramaze visual cues to enco...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
On completion of testing, rats received an overdose of sodium pentobarbital followed by intracardial perfusion with ice-cold 4% paraformaldehyde in 0.1 M PBS. Brains were remove...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Forty-µm sections were also taken from the same brains around the areas of cannula implantations to confirm placements using cresyl violet. Photomicrographs were taken usin...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
DAB was used to inhibit glycogen phosphorylase and to limit the production of lactate from glycogen in astrocytes.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Spatial working memory was assessed using spontaneous alternation tasks,,. Spontaneous alternation was chosen to assess memory because it requires no food reward and thus no...; The pharmacological manipulations were performed as within-subjects testing of multiple doses. Each testing session occurred in a new room with new extramaze visual cues to enco...; On completion of testing, rats received an overdose of sodium pentobarbital followed by intracardial perfusion with ice-cold 4% paraformaldehyde in 0.1 M PBS. Brains were remove...; Forty-µm sections were also taken from the same brains around the areas of cannula implantations to confirm placements using cresyl violet. Photomicrographs were taken usin....
from paperStatistical comparison
DAB was used to inhibit glycogen phosphorylase and to limit the production of lactate from glycogen in astrocytes. DAB injections into the hippocampus significantly impaired alt...; Either a glucose- or a lactate-sensitive biosensor was inserted into the dorsal hippocampus via a guide cannula (Pinnacle Technology Inc., Lawrence, KS). The biosensor projected...; In this example, the left hemisphere received infusions of 1% DMSO in saline (left) and the right hemisphere received infusions of 30 pmol 4-CIN. There were no significant diffe...; All statistical analyses were done using SPSS v. 18.0 (SPSS, Chicago, IL). For each experiment, repeated measures ANOVAs were used to analyze differences. Fisher's least signifi...
from paperReporting output
Report representative outputs alongside summary comparisons for Spatial working memory was assessed using spontaneous alternation tasks,,. Spontaneous alternation was chosen to assess memory because it requires no food reward and thus no..., The pharmacological manipulations were performed as within-subjects testing of multiple doses. Each testing session occurred in a new room with new extramaze visual cues to enco..., On completion of testing, rats received an overdose of sodium pentobarbital followed by intracardial perfusion with ice-cold 4% paraformaldehyde in 0.1 M PBS. Brains were remove..., Forty-µm sections were also taken from the same brains around the areas of cannula implantations to confirm placements using cresyl violet. Photomicrographs were taken usin....
inferred from protocolStructured statistical methods
DAB was used to inhibit glycogen phosphorylase and to limit the production of lactate from glycogen in astrocytes. DAB injections into the hippocampus significantly impaired alt...; Either a glucose- or a lactate-sensitive biosensor was inserted into the dorsal hippocampus via a guide cannula (Pinnacle Technology Inc., Lawrence, KS). The biosensor projected...; In this example, the left hemisphere received infusions of 1% DMSO in saline (left) and the right hemisphere received infusions of 30 pmol 4-CIN. There were no significant diffe...; All statistical analyses were done using SPSS v. 18.0 (SPSS, Chicago, IL). For each experiment, repeated measures ANOVAs were used to analyze differences. Fisher's least signifi...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Male, Sprague-Dawley rats (Harlan Laboratories; 3 months old at the time of surgery) were housed individually with free access to food and water and maintained on a 12∶12 hr light/dark cycle with lights on at 7:00 am. All procedures described in this paper were approved by the University of Illinois Urbana-Champaign Institutional Animal Care and Use Committee in accordance with guidelines outlined in Guide for Care and Use of Laboratory Animals and accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.
Rats were anesthetized with isoflurane and placed in a stereotaxic frame. In studies of the effects on memory of intrahippocampal drug infusions, two 8-mm guide cannulae (Plastics One) were chronically implanted bilaterally above the central portion of the ventral hippocampus (coordinates: 5.5 mm posterior and ±4.8 mm lateral from bregma and 4.2 mm ventral from skull) to accommodate infusion cannulae near the time of behavioral testing. Due to the shape and size of the housing for the wireless potentiostat used for measurements of changes in extracellular glucose and lactate levels during behavioral testing, the guide cannula for bioprobes needed to be placed close to the midline of the skull. Therefore the guide cannulae for bioprobes were implanted above the dorsal hippocampus instead of the ventral hippocampus in either the left or right hemisphere (coordinates: 3.8 mm posterior and 2.5 mm lateral from bregma and 1.9 mm ventral from skull). We have previously found that direct infusions of glucose into either the dorsal or ventral, hippocampus were both effective in enhancing memory during spontaneous alternation testing. All rats were allowed at least 1 week to recover...
Spatial working memory was assessed using spontaneous alternation tasks,,. Spontaneous alternation was chosen to assess memory because it requires no food reward and thus no food restriction; therefore the natural levels of glucose or lactate would be at baseline at the start of testing. The task was also chosen because it measures spatial working memory, which is sensitive to hippocampal manipulations. In the current experiments, animals were placed on a four-arm, plus-shaped maze (arms: 45 cm long, 14 cm wide, 7.5 cm tall; center area: 14 × 14 cm) constructed of opaque, black Plexiglas, as described previously,, or in a four-arm, plus-shaped maze with slightly higher sides (45 cm long, 14 cm wide, and 15 cm tall; center area: 14 × 14 cm) that were made of clear Plexiglas. The maze with higher arms was used to contain the rats better on the maze in experiments in which a delay was imposed between arm choices,. The maze was located in the center of the testing rooms on a table 76 cm above the floor surrounded by a rich assortment of extra-maze visual cues. During each testing session, the rat was placed in a start arm and allowed to explore the maze freely for 20 mi...
Either a glucose- or a lactate-sensitive biosensor was inserted into the dorsal hippocampus via a guide cannula (Pinnacle Technology Inc., Lawrence, KS). The biosensor projected 3 mm beyond the end of the implanted cannula. The last 1 mm of the probe was coated with lactate oxidase or glucose oxidase to metabolize the respective analyte, generating a current measured by the probe. The biosensor was connected to a potentiostat inside the head cap, which sent readings of the current generated by lactate or glucose in extracellular fluid to a computer by telemetry, with data recording and storage in 1-sec bins accomplished with Pinnacle Technology Laboratory v. 1.6.7 software. Glucose biosensors have previously been shown to have a range of 0-10 mM with in vitro sensitivity of 1.6±0.4 nA/mM (mean ± SEM) and lactate biosensors have a range of 0-8 mM with an in vitro sensitivity of 4.6±0.6 nA/mM (mean ± SEM),. To confirm the accuracy of the biosensors prior to implantation and immediately following testing, the probe was placed in 0.1 M PBS, connected to the potentiostat, and readings were allowed to stabilize (generally stable within 15-30 min...
All injections (0.5 µl, 0.9% saline vehicle, pH = 7.2) were made bilaterally into the ventral hippocampus with a CMA/100 microinjection pump at a flow rate of 0.25 µl/min 5 min prior to behavioral testing. Ten rats were injected with lactate (0, 50, 100, or 150 nmol of lactate in 0.5 µl of 0.9% saline, pH = 7.2), 12 rats were injected with 1,4-dideoxy-1,4-imino-D-aribinitol (DAB; 0, 0.5, 5, 50 pmol, 50 pmol+25 nmol glucose, or 50 pmol+50 nmol lactate in 0.5 µl of 0.9% saline, pH = 7.2), and 8 rats were injected with α-cyano-4-hydroxycinnamate (4-CIN; 3, 10, 30 pmol, 30 pmol+25 nmol glucose, or 30 pmol+50 nmol lactate in 0.5 µl of 1% DMSO and 0.9% saline, pH = 7.2) were infused into the hippocampus 5 min prior to behavioral testing. DAB, an inhibitor of glycogen phosphorylase, was used to block glycogenolysis in astrocytes,,. Low doses of 4-CIN were used to block the neuronal transporter, MCT2, while not blocking astrocytic MCT1 or MCT4 transporters,,,,,,,. Doses were given in a counterbalanced order using a Latin squares design with each main treatment tested in separate groups of rats. In t...
On completion of testing, rats received an overdose of sodium pentobarbital followed by intracardial perfusion with ice-cold 4% paraformaldehyde in 0.1 M PBS. Brains were removed and postfixed overnight in 4% paraformaldehyde. They were then transferred to a 20% glycerol solution until they lost buoyancy (∼48 hrs). They were then sectioned on a cryostat (Leica CM1850, Leica Microsystems Inc., Germany) with 20 µm slices of prefrontal cortex, striatum, dorsal hippocampus, and ventral hippocampus saved for glycogen localization analysis using a periodic acid-Schiff's reagent method (PAS) previously demonstrated to highlight glycogen. Prior to PAS staining, immunofluorescence methods were used to stain neurons with neuronal nuclear antigen (NeuN) and astrocytes with glial fibrillary acidic protein (GFAP) so that all sections were stained with PAS and consecutive sections were stained for NeuN and GFAP. Briefly, for GFAP immunofluorescence, the tissue was rinsed in 0.05 M PBS 4 × for 10 minutes and then incubated in a 5% normal goat serum (NGS), 1% bovine serum albumin (BSA), and 0.2% Triton X-100 in 0.05 M PBS for 60 minutes to block endogenous proteins. Next, the ti...
To show that 4-CIN did not block monocarboxylate transport into mitochondria, activity of the mitochondria was monitored using a stain for succinate dehydrogenase (SDH) activity,. SDH activity has previously been demonstrated to decrease in the presence of 3-bromopyruvate and with sensory deprivation,, supporting a relationship between SDH activity and mitochondrial function. Prior to sacrifice, the rats used in the 4-CIN dose response experiment received 30 pmol 4-CIN unilaterally in the ventral hippocampus and 1% DMSO in saline in the other hemisphere, providing within-subjects comparisons. The hemispheres receiving 4-CIN were counterbalanced across rats. Five min after the injection animals were tested on spontaneous alternation for 20 minutes to parallel the other behavioral experiments. The rats then received an overdose of sodium pentobarbital and were perfused transcardially with 10% glycerol and 0.5% paraformaldehyde in PBS. The brains were removed, blocked, frozen rapidly in heptane cooled with dry ice, and sectioned in the coronal plane at 30 µm in a cryostat. Alternating 40 µm sections were saved from ventral hippocampus for cresyl violet nissl staining...
All statistical analyses were done using SPSS v. 18.0 (SPSS, Chicago, IL). For each experiment, repeated measures ANOVAs were used to analyze differences. Fisher's least significant difference post hoc tests were conducted when the results from the ANOVAs were significant.
Machine-readable layer
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"name": "Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing methods",
"description": "Evidence-backed execution summary for Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing methods from Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing.",
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"text": "Male, Sprague-Dawley rats (Harlan Laboratories; 3 months old at the time of surgery) were housed individually with free access to food and water and maintained on a 12∶12 hr light/dark cycle with lights on at 7:00 am. All procedures described in this paper were approved by the University of Illinois Urbana-Champaign Institutional Animal Care and Use Committee in accordance with guidelines outlined in Guide for Care and Use of Laboratory Animals and accredited by the Association for Assessment and Accreditation of Laboratory Animal Care."
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"text": "Rats were anesthetized with isoflurane and placed in a stereotaxic frame. In studies of the effects on memory of intrahippocampal drug infusions, two 8-mm guide cannulae (Plastics One) were chronically implanted bilaterally above the central portion of the ventral hippocampus (coordinates: 5.5 mm posterior and ±4.8 mm lateral from bregma and 4.2 mm ventral from skull) to accommodate infusion cannulae near the time of behavioral testing. Due to the shape and size of the housing for the wireless potentiostat used for measurements of changes in extracellular glucose and lactate levels during behavioral testing, the guide cannula for bioprobes needed to be placed close to the midline of the skull. Therefore the guide cannulae for bioprobes were implanted above the dorsal hippocampus instead of the ventral hippocampus in either the left or right hemisphere (coordinates: 3.8 mm posteri..."
},
{
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"name": "Memory testing",
"text": "Spatial working memory was assessed using spontaneous alternation tasks,,. Spontaneous alternation was chosen to assess memory because it requires no food reward and thus no food restriction; therefore the natural levels of glucose or lactate would be at baseline at the start of testing. The task was also chosen because it measures spatial working memory, which is sensitive to hippocampal manipulations. In the current experiments, animals were placed on a four-arm, plus-shaped maze (arms: 45 cm long, 14 cm wide, 7.5 cm tall; center area: 14 × 14 cm) constructed of opaque, black Plexiglas, as described previously,, or in a four-arm, plus-shaped maze with slightly higher sides (45 cm long, 14 cm wide, and 15 cm tall; center area: 14 × 14 cm) that were made of clear Plexiglas. The maze with higher arms was used to contain the rats better on the maze in experiments in which a d..."
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{
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"name": "Bioprobe measurements of extracellular hippocampal glucose and lactate during behavioral testing",
"text": "Either a glucose- or a lactate-sensitive biosensor was inserted into the dorsal hippocampus via a guide cannula (Pinnacle Technology Inc., Lawrence, KS). The biosensor projected 3 mm beyond the end of the implanted cannula. The last 1 mm of the probe was coated with lactate oxidase or glucose oxidase to metabolize the respective analyte, generating a current measured by the probe. The biosensor was connected to a potentiostat inside the head cap, which sent readings of the current generated by lactate or glucose in extracellular fluid to a computer by telemetry, with data recording and storage in 1-sec bins accomplished with Pinnacle Technology Laboratory v. 1.6.7 software. Glucose biosensors have previously been shown to have a range of 0-10 mM with in vitro sensitivity of 1.6±0.4 nA/mM (mean ± SEM) and lactate biosensors have a range of 0-8 mM with an in vitro..."
},
{
"@type": "HowToStep",
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"name": "Intrahippocampal Injections to enhance or impair memory",
"text": "All injections (0.5 µl, 0.9% saline vehicle, pH = 7.2) were made bilaterally into the ventral hippocampus with a CMA/100 microinjection pump at a flow rate of 0.25 µl/min 5 min prior to behavioral testing. Ten rats were injected with lactate (0, 50, 100, or 150 nmol of lactate in 0.5 µl of 0.9% saline, pH = 7.2), 12 rats were injected with 1,4-dideoxy-1,4-imino-D-aribinitol (DAB; 0, 0.5, 5, 50 pmol, 50 pmol+25 nmol glucose, or 50 pmol+50 nmol lactate in 0.5 µl of 0.9% saline, pH = 7.2), and 8 rats were injected with α-cyano-4-hydroxycinnamate (4-CIN; 3, 10, 30 pmol, 30 pmol+25 nmol glucose, or 30 pmol+50 nmol lactate in 0.5 µl of 1% DMSO and 0.9% saline, pH = 7.2) were infused into the hippocampus 5 min prior to behavioral testing. DAB, an inhibitor of glycogen phosphorylase, was used to block glycogenolysis..."
},
{
"@type": "HowToStep",
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"name": "Histology for glycogen localization and for cannula placements",
"text": "On completion of testing, rats received an overdose of sodium pentobarbital followed by intracardial perfusion with ice-cold 4% paraformaldehyde in 0.1 M PBS. Brains were removed and postfixed overnight in 4% paraformaldehyde. They were then transferred to a 20% glycerol solution until they lost buoyancy (∼48 hrs). They were then sectioned on a cryostat (Leica CM1850, Leica Microsystems Inc., Germany) with 20 µm slices of prefrontal cortex, striatum, dorsal hippocampus, and ventral hippocampus saved for glycogen localization analysis using a periodic acid-Schiff's reagent method (PAS) previously demonstrated to highlight glycogen. Prior to PAS staining, immunofluorescence methods were used to stain neurons with neuronal nuclear antigen (NeuN) and astrocytes with glial fibrillary acidic protein (GFAP) so that all sections were stained with PAS and consecutive sections were..."
},
{
"@type": "HowToStep",
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"name": "Histology for glycogen localization and for cannula placements",
"text": "To show that 4-CIN did not block monocarboxylate transport into mitochondria, activity of the mitochondria was monitored using a stain for succinate dehydrogenase (SDH) activity,. SDH activity has previously been demonstrated to decrease in the presence of 3-bromopyruvate and with sensory deprivation,, supporting a relationship between SDH activity and mitochondrial function. Prior to sacrifice, the rats used in the 4-CIN dose response experiment received 30 pmol 4-CIN unilaterally in the ventral hippocampus and 1% DMSO in saline in the other hemisphere, providing within-subjects comparisons. The hemispheres receiving 4-CIN were counterbalanced across rats. Five min after the injection animals were tested on spontaneous alternation for 20 minutes to parallel the other behavioral experiments. The rats then received an overdose of sodium pentobarbital and were perfused transcardiall..."
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"name": "Statistical Analysis",
"text": "All statistical analyses were done using SPSS v. 18.0 (SPSS, Chicago, IL). For each experiment, repeated measures ANOVAs were used to analyze differences. Fisher's least significant difference post hoc tests were conducted when the results from the ANOVAs were significant."
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"name": "Bioprobe measurements of extracellular hippocampal glucose and lactate during behavioral testing"
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"headline": "Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing",
"datePublished": "2011",
"author": [
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"@type": "Person",
"name": "Lori A. Newman"
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"name": "Donna L. Korol"
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"identifier": "10.1371/journal.pone.0028427"
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