This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer’s disease.
The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer’s disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in “healthy” and “unhealthy” slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.
1. Preparing Ice-cold Oxygenated Artificial Cerebrospinal Fluid (ACSF)
2. Brain Removal and Hippocampal Dissection in Aged (> 20-month-old-rats)
3. Brain Removal and Hippocampal Dissection in Aged Transgenic Mice
4. Section Brain Tissue Into Slices using a Vibrating Microtome (Vibratome) and Transfer to Holding Chamber*
5. Elicit and Record CA3-CA1 Synaptic Responses
6. Representative Results
Our work, and work from other groups, suggests that changes in astrocyte-based inflammatory signaling may trigger and/or hasten neurologic dysfunction during aging and AD 13,20,21. Recently, we have used synaptic strength, LTP, and LTD as endpoint measures to investigate the efficacy and mechanisms of action of several novel anti-inflammatory reagents in mid-aged APP/PS1 mice (see 22 for description of this model) and aged Fischer 344 rats. The results provided below were obtained using the protocols described in this article.
One of the novel anti-inflammatory adeno-associated viral (AAV) reagents developed by our lab has been shown in pilot studies to significantly increase synaptic strength (p < 0.05) and prevent LTP deficits (p < 0.05) in mid-aged (16-month-old) APP/PS1 mice (n = 4-6 slices per treatment condition). Representative synaptic strength curves and LTP experiments from two different slices, collected from the same 16-month-old APP/PS1 mouse, are shown in Figure 5A-C. One slice was extracted from the hemisphere treated with our novel AAV (Reagent A), while the other slice was treated with a control AAV reagent (Control). LTP was induced in both slices using two 1 sec trains of 100 Hz stimulation (10 sec intertrain interval). Note that the synaptic strength curve for the Reagent A-treated slice is shifted to the left of the control slice, indicative of greater synaptic strength. Also note that, typical for mid-aged APP/PS1 mice, LTP decayed rapidly to baseline in the control slice (e.g.23). Conversely, LTP decayed little in the slice treated with our novel reagent.
In a second recent study, we observed significant LTD in vehicle-treated aged rats (85 % of pre-LTD baseline, p < 0.05). In contrast, no LTD was observed in aged rats treated with novel anti-inflammatory “Drug A” (97 % of pre-LTD baseline, not significant). No drug effects on synaptic strength were observed. Representative LTD experiments from this data set (n = 8-10 rats per group) are illustrated in Figure 5D-F.
Figure 1. Tools and materials used for brain dissection. A, paper towel. B, scalpel blade. C, Beebee scissors. D, bone rongeurs (for rats). E, bone rongeurs (for mice/rats). F, Plastic spoon. G, plastic transfer pipette. H, Hippocampal tool. I, spatula. J, surgical scissors. K, glass petri dish.
Figure 2. Custom brain slice holding chamber. A, macrochamber. B, lid. C, H2O reservoir with perforated silicone tubing. D, microchamber. E, ACSF delivery tube (polyethylene). F, O2 delivery tube. G, port for temperature control. H, netted microchamber insert.
Figure 3. RC22 submersion chamber. A, Recording chamber. B, Ground electrode. C, Aspiration needle.
Figure 4. Hippocampal slice illustration and extracellular waveforms. A, Cartoon of a transverse hippocampal section used in electrophysiology experiments. CA = cornu Ammonis. DG = dentate gyrus. SC = Schaffer collaterals. S radiatum = stratum radiatum. B, Electrical stimulation of the SC (a CA3 axon tract) elicits a stimulus artifact, followed almost immediately by a presynaptic population spike, or fiber volley (FV). The amplitude of FV is directly proportional to the number of SC fibers activated. The slope of the negative-going phase of the field excitatory postsynaptic potential (EPSP) corresponds directly to the activation of depolarizing synaptic currents in CA1 pyramidal neurons in response to glutamate release from SC terminals. C, Overlapping representative extracellular waveforms recorded in CA1 stratum radiatum in response to nine different stimulus intensity levels (30-500 μA) in a “healthy” (left panel), “unhealthy”, and “hyperexcitable” slice. Five waveforms were averaged per level. Healthy slices respond dynamically across this stimulus range and exhibit a single positive-going population spike (reflecting CA1 neuronal discharge) at the higher stimulus levels. In the RC22 submersion chamber, maximal EPSPs typically range from 1.5 to 3 mV in amplitude. Unhealthy slices (middle panel) often exhibit a large FV, but a small maximal EPSP (< 1 mV) and usually show poor plasticity. Hyperexcitable slices (right panel) show two or more regenerative population spikes in the ascending limb of the EPSP. Responses in hyperexcitable slices are often labile and are variably affected by LTD/LTP stimulation.
Figure 5. Representative electrophysiology experiments performed on acute slices from mid-aged (16 mos) APP/PS1 mice and aged (22 mos) Fisher 344 rats. Panels A-B show data collected from APP/PS1 mice treated with a control adeno-associated (AAV) viral construct (Control) or a novel AAV reagent (Reagent A) that has been developed by our lab group. Relative to the control slice, the slice treated with Reagent A exhibits a marked leftward shift in the EPSP:FV curve (A) indicative of greater synaptic strength. The Reagent-A-treated slice also shows robust and stable LTP (B) after the delivery of two 1 sec, 100 Hz stimulus trains, while the control slice exhibits deficient LTP, typical of this animal model. Panels D-F show data collected from two individual aged rats that received chronic (4 week) intrahippocampal perfusions of vehicle or a novel anti-inflammatory drug (Drug A). Basal synaptic strength was relatively unaffected by drug treatment (D). However, Drug A was very effective at preventing the induction of LTD (E). Panels C and F show representative EPSP waveforms recorded from individual slices before (pre) and 60 min after (post) the delivery of LTP/LTD stimulation. Note that stimulus artifacts are not shown.
The steps outlined in this protocol will help insure that brain dissections are carried out at least as rapidly and efficiently in aged, as in young adult rats. We also provide sufficient detail for the beginner to set up their own slice studies on LTP and LTD. If further exploration of aging and AD changes in synaptic function and plasticity is one of your goals, there are at least two other methodological issues, alluded to above, that deserve further consideration. First, several labs have shown that the Ca2+:Mg2+ ratio in recording ACSF can have a marked effect on the induction synaptic plasticity in hippocampal slices2,10,24,25. In mammalian CSF, the Ca2+:Mg2+ ratio is approximately one (e.g. see 26). However, ACSF Ca2+:Mg2+ ratios closer to 2 are commonly used in slice studies of synaptic function and plasticity. In early studies, this practice was probably adapted to optimize induction of LTP, then subsequently became routine for all plasticity studies. However, this practice can be problematic in aging and AD studies because of well-characterized differences in neuronal Ca2+ regulation. Specifically, Ca2+ influx and/or Ca2+-induced Ca2+-release is elevated in aged rats and/or AD model mice during neuronal activation3,27-31. Induction of LTD is particularly sensitive to subtle changes in ACSF Ca2+ levels. Our protocol, which uses 2mM Ca2+ and 2 mM Mg2+, commonly results in LTD for aged, but not young adult animals2, while studies using a Ca2+:Mg2+ ratio closer to two, have observed robust LTD in adults in the absence of an age difference2,10 or in conjunction with reduced LTD in aged rats32. These observations highlight the need to carefully consider ACSF Ca2+ and Mg2+ levels when comparing Ca2+-dependent plasticity in aged and young adult animals.
The second methodological issue concerns the strong dependency of LTP on postsynaptic depolarization33 and possible aging/genotype differences in synaptic strength. In a typical LTP experiment, baseline and LTP stimulation intensity is generally adjusted to produce a half maximal (or three-quarter maximal) EPSP amplitude. The potential problem is that aged rats and APP/PS1 mice usually show reduced synaptic strength relative to their younger and/or wild type counterparts, meaning that baseline EPSP values will also be smaller in aged rats and APP/PS1 mice. Smaller EPSPs may translate to less depolarization during LTP stimulation, resulting in a reduced probability for inducing LTP33. Because of this potential confound, it is difficult to determine whether these animals exhibit a throughput deficit, a plasticity deficit or both. That is, LTP induction mechanisms in aged and/or APP/PS1 mice may be functionally intact (no plasticity deficit), but insufficiently stimulated (throughput deficit) under these conditions. This distinction is critical, as mechanisms for throughput and mechanisms for plasticity may respond very differently to a specific pharmacologic treatment. We try to minimize the impact of reduced throughput on LTP induction by normalizing the EPSP amplitude to the same level (e.g. 1 mV) across all slices prior to LTP stimulation. Other strategies may be effective as well (e.g. use of voltage or current clamp to equalize the membrane potential across groups during LTP stimulation), and should be considered when investigating LTP in these animal models.
The authors have nothing to disclose.
Work supported by NIH grant AG027297, an award from the Kentucky Spinal Cord and Head Injury Research Trust, and a gift from the Kleberg Foundation.
Name of the reagent | Company | Catalogue number |
NaCl | Fisher | BP358-1 |
KCl | Fisher | BP366-500 |
KH2PO4 (monobasic) | Sigma | P5379-100G |
MgSO4 | Sigma | M2643-500G |
CaCl2 (dihydrate) | Sigma | C3306-250G |
NaHCO3 | Fisher | S233-500 |
C6H12O6 (dextrose) | Fisher | BP350-1 |
Table 1. Reagents required
Name of the equipment | Company | Catalogue number | Yorumlar |
Erlenmeyer Flasks | Fisher | FB-500-2000 FB-500-1000 |
|
Aquarium Bubbler | Used for oxygenating media. Available at most pet stores | ||
50 mL Glass beaker | Fisher | 02-540G | For brain storarge in ACSF |
Parafilm | Fisher | 13-374-10 | |
Small Animal Guillotine | World Precision Instruments (WPI) | DCAP-M | |
Flat paper towel | |||
#11 Feather surgical blade | Fisher | 08-916-5B | |
Beebee bone scissors | Fine Science Tools (FST) | 16044-10 | |
Lempert Rongeurs | Roboz | RS-8321 | Use for rats |
Friedman-Pearson Rongeurs | FST | 16020-14 | Use for mice or rats |
Hippocampus tool | FST | 10099-15 | |
Spoon | A plastic teaspoon will do | ||
Spatula | Fisher | 21-401-25A | Spatula |
Surgical iris scissors | FST | 14058-09 | |
plastic transfer pipets | Fisher | 13-711-43 | |
110mm Whatman filter paper | Fisher | 09-805E | Whatman cat. 1001-110 |
Glass petri dish | Fisher | ||
Leica VT1000P Manual Vibrating Microtome | Vibratome | VT1000P | |
0.1mm FA-10 Feather S blade | Ted Pella | 121-9 | 0.1mm FA-10 Feather S blade |
Borosilicate Glass Pasteur Pipet (with rubber bulb) | Fisher | 13-678-20A | For transferring slices: Tip is broken off and heat-polished for larger opening |
35 mm Polysterine Culture dish | Corning | 430588 | Used for collecting slices after dissection |
Table 2. Tools and materials for dissection
Equipment/Materials | Company | Comments (optional) |
Holding chamber | Custom Built | |
P-97 Horizontal Pipette Puller | Sutter Instrument Co. | |
Vibration isolation table | Technical Manufacturing Corporation (TMC) | |
Faraday cage | Custom built | |
Pyrex Aspirator Bottle with Bottom Sidearm (Product #1220-1L) | Corning | |
Gravity-contolled IV set with regulator (Product # 2C8891) | Baxter | |
Central Vacuum Line | Available in most modern labs | |
95% O2 / 5% CO2 Gas Mix | Scott-Gross Co. | |
TygonTM Lab tubing For O2/CO2 delivery |
Fisher Scientific | Non-toxic, non-oxidizing, comes in a variety of sizes. |
Eclipse E600FN Microscope | Nikon | with 10x and 40x objectives, near infared filter, and GFP,DS-Red2 filters |
Cool Snap ES Digital Camera | Photometrics | Cool Snap ES Digital Camera |
X-Cite Fluorescent Illuminator | EXFO | X-Cite Fluorescent Illuminator |
Microscope Platform | Siskiyou | Custom assembled |
RC-22 Submersible recording chamber (Product # 64-0228) | Warner Instruments (WI) | Requires P-1 platform and stage adaptor (Product # 64-0277 From Warner) |
TC2BIP 2/3Ch Temperature controller | Cell Microcontrols | TC2BIP 2/3Ch Temperature controller |
4 Axis Manual Miniature manipulator | Siskiyou | |
Platinum Iridium Wire (0.002 in) (item # PTT0203) |
WPI | |
A365 Stimulus Isolator | WPI | A365 Stimulus Isolator |
Multiclamp 700b Amplifier | Axon Instruments | |
Digidata 1322A A/D converter | Axon Instruments | |
PClamp software | Axon Instruments | |
Personal Computer (Pentium 4) | Dell |
Table 3. Electrophysiology equipment and materials