Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice methods
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Parabiosis
Parabiosis surgery followed previously described procedures,. Mirror-image incisions at the left and right flanks were made through the skin, and shorter incisions were made through the abdominal wall. The peritoneal openings of the adjacent parabionts were sutured together. Elbow and knee joints from each parabiont were sutured together, and the skin of each mouse was stapled (9-mm Autoclip, Clay Adams) to the skin of the adjacent parabiont. Each mouse was injected subcutaneously with enrofloxacin (Bayer) antibiotic and buprenorphine (Butler Schein) as directed for pain and monitored during recovery. For overall health and maintenance behavior, several recovery characteristics were analyzed at various times after surgery, including paired weights, grooming and stress responses. General appearance was recorded at the indicated intervals using a basic grooming appearance scale that r...
Stereotaxic injections
Animals were placed in a stereotaxic frame and anesthetized with 2% isoflurane (Patterson Veterinary) (2 l per min oxygen flow rate) delivered through an anesthesia nose cone. Ophthalmic eye ointment was applied to the cornea to prevent desiccation during surgery. The area around the incision was trimmed. Viral solutions were injected bilaterally into the dorsal hippocampi using the following coordinates: (from bregma) anterior = -2 mm, lateral = 1.5 mm and (from skull surface) height = -2.1 mm. A 2 µl volume was injected stereotaxically over 10 min (injection speed: 0.20 µl per min) using a 5-µl 26s-gauge Hamilton syringe. To limit reflux along the injection track, the needle was maintained in situ for 8 min, slowly pulled out half-way and kept in position for an additional 2 min. The skin was closed using silk suture. Each mouse was injected subcutaneously...
Golgi staining
After brain removal, hemispheres were immersed in 10 ml modified Golgi-Cox staining solution (courtesy of D. Jing and F. Lee at Cornell University) for 7-10 d at room temperature in the dark. Brains were transferred to 30% sucrose in dH 2 O at 4 °C for 72 h. Sections (150 µm) were mounted onto slides coated with 0.3% gelatin in dH 2 O. After briefly drying, slides were dipped in 40% sucrose three times and air dried for 72 h in the dark. Slides were immersed into dH 2 O three times for 10 min with gentle shaking and then transferred to a developing solution for 6 min. Slides were then rinsed three times for 10 min in dH 2 O, dehydrated through graded ethanol, immersed in Histoclear and then coverslipped using DPX mounting medium (Sigma-Aldrich). Neurons were traced at 100 ×, and all subsequent analyses were done using Neurolucida Software (v10, MBF Bioscience). Sho...
Extracellular electrophysiology
Extracellular electrophysiology was performed as previously described. Acute hippocampal slices (400-µm thick) were prepared from aged parabionts. Slices were maintained in artificial cerebrospinal fluid (ACSF; (in mM) NaCl, 124.0; KCl, 2.5; KH 2 PO4, 1.2; CaCl 2, 2.4; MgSO 4, 1.3; NaHCO 3, 26.0; and glucose, 10.0) continuously oxygenated with 5% CO 2 and 95% O 2. Recordings were performed with an Axopatch-2B amplifier and pClamp 10.2 software (Axon Instruments). Submerged slices were continuously perfused with oxygenated ACSF at a flow rate of 2 ml per min from a reservoir by gravity feeding. Field potential (population spikes) was recorded using glass microelectrodes filled with ACSF (resistance: 4-8 MΩ). Biphasic current pulses (0.2-ms duration for one phase, 0.4 ms in total) were delivered in 10-s intervals through a concentric bipolar stimulating electrode (F...
Western blot analysis
Mouse hippocampi were dissected after perfusion of the animal, snap frozen and lysed in RIPA lysis buffer (500 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Na deoxycholate, 1% NP40, 0.1% SDS and cOmplete protease inhibitors; Roche). Tissue lysates were mixed with 4 × NuPage LDS loading buffer (Invitrogen), loaded on a 4-12% SDS polyacrylamide gradient gel (Invitrogen) and subsequently transferred onto a nitrocellulose membrane. The blots were blocked in 5% milk in Tris-buffered saline with Tween (TBST) and incubated with rabbit anti-actin (1:5,000, A-5060, Sigma), mouse anti-Gapdh (1:10,000, 6C5, Abcam) rabbit anti-Creb (1:1,000, 48H2, Cell Signaling) and rabbit anti-pCreb (1:1,000, Ser133, D1G6, Cell Signaling). Horseradish peroxidase-conjugated secondary antibodies and an ECL kit (GE Healthcare/Amersham Pharmacia Biotech) were used to detect protein signals. Multiple exposures...
Contextual fear conditioning
Our paradigm followed previously published techniques. Mice learned to associate the environmental context (fear-conditioning chamber) with an aversive stimulus (mild foot shock; unconditioned stimulus, US), which enabled testing for hippocampal-dependent contextual fear conditioning. The mild foot shock was paired with a light and tone cue (conditioned stimulus, CS) in order to also assess amygdaladependent cued fear conditioning. Conditioned fear was displayed as freezing behavior. Specific training parameters were as follows: tone duration, 30 s; level, 70 dB, 2 kHz; shock duration, 2 s; intensity, 0.6 mA. On day 1, each mouse was placed in a fear-conditioning chamber and allowed to explore for 2 min before delivery of a 30-s tone (70 dB) ending with a 2-s foot shock (0.6 mA). Two minutes later, a second CS-US pair was delivered. On day 2, each mouse was first placed in the fear-c...
Plasma collection
Pooled mouse plasma was collected from 700 young (3 months) or aged (18 months) mice by intracardial bleed at time of euthanasia. Plasma was prepared from blood collected with EDTA followed by centrifugation at 1,000 g. For plasma denaturation, plasma was heated for 2-3 min at 95 °C, followed by a short spin at 1,000 g. All plasma aliquots were stored at -80 °C until use. Before administration, plasma was dialyzed using 3.5-kDa D-tube dialyzers (EMD Millipore) in PBS to remove EDTA. Young adult mice were systemically treated with plasma (100 µl per injection) isolated from young or aged mice by intraorbital injections or intravenously into the tail vein eight times over 24 d.
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Evidence quotes (7)
Parabiosis surgery followed previously described procedures,. Mirror-image incisions at the left and right flanks were made through the skin, and shorter incisions were made through the abdominal wall. The peritoneal openings of the adjacent parabionts were sutured together. Elbow and knee joints from each parabiont were sutured together, and the skin of each mouse was stapled (9-mm Autoclip, Clay Adams) to the skin of the adjacent parabiont. Each mouse was injected subcutaneously with enrofloxacin (Bayer) antibiotic and buprenorphine (Butler Schein) as directed for pain and monitored during recovery. For overall health and maintenance behavior, several recovery characteristics were analyzed at various times after surgery, including paired weights, grooming and stress responses. General appearance was recorded at the indicated intervals using a basic grooming appearance scale that ranged from an absence of grooming behavior (0) to normal, healthy grooming behavior (4). Intermediate scores reflect the degree and frequency at which fur was groomed and/or maintained on various regions of the paired body, including muzzle, eyes, head, torso and legs, adapted from guidelines from S...
Animals were placed in a stereotaxic frame and anesthetized with 2% isoflurane (Patterson Veterinary) (2 l per min oxygen flow rate) delivered through an anesthesia nose cone. Ophthalmic eye ointment was applied to the cornea to prevent desiccation during surgery. The area around the incision was trimmed. Viral solutions were injected bilaterally into the dorsal hippocampi using the following coordinates: (from bregma) anterior = -2 mm, lateral = 1.5 mm and (from skull surface) height = -2.1 mm. A 2 µl volume was injected stereotaxically over 10 min (injection speed: 0.20 µl per min) using a 5-µl 26s-gauge Hamilton syringe. To limit reflux along the injection track, the needle was maintained in situ for 8 min, slowly pulled out half-way and kept in position for an additional 2 min. The skin was closed using silk suture. Each mouse was injected subcutaneously with the analgesic buprenorphine (Butler Schein). Mice were single housed and monitored during recovery.
After brain removal, hemispheres were immersed in 10 ml modified Golgi-Cox staining solution (courtesy of D. Jing and F. Lee at Cornell University) for 7-10 d at room temperature in the dark. Brains were transferred to 30% sucrose in dH 2 O at 4 °C for 72 h. Sections (150 µm) were mounted onto slides coated with 0.3% gelatin in dH 2 O. After briefly drying, slides were dipped in 40% sucrose three times and air dried for 72 h in the dark. Slides were immersed into dH 2 O three times for 10 min with gentle shaking and then transferred to a developing solution for 6 min. Slides were then rinsed three times for 10 min in dH 2 O, dehydrated through graded ethanol, immersed in Histoclear and then coverslipped using DPX mounting medium (Sigma-Aldrich). Neurons were traced at 100 ×, and all subsequent analyses were done using Neurolucida Software (v10, MBF Bioscience). Sholl analysis was performed for each neuron by placing concentric spheres at 10-µm intervals from the soma. The number of times the dendrite intersected each sphere and the total dendritic length within each sphere were quantified. Dendritic length was summed across distance in the x, y, and z...
Extracellular electrophysiology was performed as previously described. Acute hippocampal slices (400-µm thick) were prepared from aged parabionts. Slices were maintained in artificial cerebrospinal fluid (ACSF; (in mM) NaCl, 124.0; KCl, 2.5; KH 2 PO4, 1.2; CaCl 2, 2.4; MgSO 4, 1.3; NaHCO 3, 26.0; and glucose, 10.0) continuously oxygenated with 5% CO 2 and 95% O 2. Recordings were performed with an Axopatch-2B amplifier and pClamp 10.2 software (Axon Instruments). Submerged slices were continuously perfused with oxygenated ACSF at a flow rate of 2 ml per min from a reservoir by gravity feeding. Field potential (population spikes) was recorded using glass microelectrodes filled with ACSF (resistance: 4-8 MΩ). Biphasic current pulses (0.2-ms duration for one phase, 0.4 ms in total) were delivered in 10-s intervals through a concentric bipolar stimulating electrode (FHC, Inc.). No obvious synaptic depression or facilitation was observed with this frequency stimulation. To record field population spikes in the dentate gyrus, the recording electrode was placed in the lateral or medial side of the dorsal part of the dentate gyrus. The stimulating electrode was plac...
Mouse hippocampi were dissected after perfusion of the animal, snap frozen and lysed in RIPA lysis buffer (500 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Na deoxycholate, 1% NP40, 0.1% SDS and cOmplete protease inhibitors; Roche). Tissue lysates were mixed with 4 × NuPage LDS loading buffer (Invitrogen), loaded on a 4-12% SDS polyacrylamide gradient gel (Invitrogen) and subsequently transferred onto a nitrocellulose membrane. The blots were blocked in 5% milk in Tris-buffered saline with Tween (TBST) and incubated with rabbit anti-actin (1:5,000, A-5060, Sigma), mouse anti-Gapdh (1:10,000, 6C5, Abcam) rabbit anti-Creb (1:1,000, 48H2, Cell Signaling) and rabbit anti-pCreb (1:1,000, Ser133, D1G6, Cell Signaling). Horseradish peroxidase-conjugated secondary antibodies and an ECL kit (GE Healthcare/Amersham Pharmacia Biotech) were used to detect protein signals. Multiple exposures were taken to select images within the dynamic range of the film (GE Healthcare Amersham Hyperfilm ECL). Selected films were scanned (300 dots per inch) and quantified using ImageJ software (Version 1.46k). Actin bands were used for normalization.
Our paradigm followed previously published techniques. Mice learned to associate the environmental context (fear-conditioning chamber) with an aversive stimulus (mild foot shock; unconditioned stimulus, US), which enabled testing for hippocampal-dependent contextual fear conditioning. The mild foot shock was paired with a light and tone cue (conditioned stimulus, CS) in order to also assess amygdaladependent cued fear conditioning. Conditioned fear was displayed as freezing behavior. Specific training parameters were as follows: tone duration, 30 s; level, 70 dB, 2 kHz; shock duration, 2 s; intensity, 0.6 mA. On day 1, each mouse was placed in a fear-conditioning chamber and allowed to explore for 2 min before delivery of a 30-s tone (70 dB) ending with a 2-s foot shock (0.6 mA). Two minutes later, a second CS-US pair was delivered. On day 2, each mouse was first placed in the fear-conditioning chamber containing the same exact context but with no administration of a CS or foot shock. Freezing was analyzed for 1-3 min. One hour later, the mice were placed in a new context containing a different odor, cleaning solution, floor texture, chamber walls and shape. Animals were...
Pooled mouse plasma was collected from 700 young (3 months) or aged (18 months) mice by intracardial bleed at time of euthanasia. Plasma was prepared from blood collected with EDTA followed by centrifugation at 1,000 g. For plasma denaturation, plasma was heated for 2-3 min at 95 °C, followed by a short spin at 1,000 g. All plasma aliquots were stored at -80 °C until use. Before administration, plasma was dialyzed using 3.5-kDa D-tube dialyzers (EMD Millipore) in PBS to remove EDTA. Young adult mice were systemically treated with plasma (100 µl per injection) isolated from young or aged mice by intraorbital injections or intravenously into the tail vein eight times over 24 d.
Machine-readable layer
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