This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.
Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol’s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.
The advent of mammalian acute brain slice preparations facilitated experiments at the cellular and synaptic level that were previously possible only in invertebrate preparations like Aplysia1. Development of acute hippocampal slices was of particular significance, as it is a structure responsible for working memory and context formation, and has a specialized tri-synaptic circuitry that is amenable to easy physiological manipulation. However, the vast majority of acute brain slices are still prepared from relatively young mice and rats, as it is easier to preserve healthy neurons and circuits, and the slices remain viable for longer periods of time2,3,4. Here, we introduce modifications to standard slicing protocols that result in increased viability of acute hippocampal slices from adult and aging mice.
The major impediment to the long-term ex vivo viability of mammalian brain parenchyma is the initial hypoxic damage that occurs rapidly once blood flow to the brain stops following decapitation. Loss of oxygen results in fast metabolic consumption of major energy resources in the brain with the loss of phospho-creatine (P-creatine) being the most rapid, followed by glucose, adenosine triphosphate (ATP), and glycogen4. Preservation of ATP is of particular importance for the long-term health of brain slices, as ATP is needed to maintain the membrane potential via the Na-K ATPase, and consequently the neural activity5,6. The ATP level in the adult rodent brain is ~2.5 mM, and it drops precipitously within 20 s of decapitation to reach a basal steady state (~0.5 mM) at around 1 min post-decapitation4,7,8. In young animals, it takes longer to observe the same drop in ATP (~2 min); with phenobarbital anesthesia it is further slowed to 4 min4. These considerations show that preventing loss of ATP and other energy resources is a necessary strategy to prevent hypoxic damage to the brain and in turn to maintain the health of brain slices over longer periods of time, especially in adult animals.
Low temperatures slow down the metabolism. Consequently, it has been demonstrated that modest hypothermia protects brain energy reserves: in young animals, lowering body temperature by six degrees, from 37 °C to 31 °C, preserves ATP levels to around 80% of normal levels over 4 h of controlled hypoxia9. P-creatine levels are similarly preserved, as well as the overall phosphorylation potential9. This suggests that lowering body temperature prior to decapitation could be neuroprotective, as near-normal levels of ATP could be maintained through the slice cutting and slice recovery periods.
To the degree that an ATP drop cannot be completely prevented upon decapitation, a partially impaired function of the Na-K ATPase is expected, followed by depolarization via passive sodium influx. As the passive sodium influx is followed by water entry into cells, it causes cytotoxic edema and eventually pyknosis. In adult rats, replacing Na+ ions with sucrose in slice-cutting solutions has been a successful strategy to alleviate the burden of cytotoxic edema10,11. More recently, methylated organic cations that decrease sodium channel permeability12 have shown to offer more effective protection than sucrose, especially in slices from adult mice, with N-methyl-D-glucamine (NMDG) being most widely applicable across different ages and brain regions13,14,15,16.
Numerous brain-slicing protocols involve using cold temperatures only during the slice-cutting step, sometimes in combination with Na+ ion replacement strategy16,17. In young animals, these protocols appear to offer sufficient neuroprotection since the brains can be extracted quickly after decapitation because the skull is still thin and easy to remove3. However, this strategy does not produce healthy slices from adult animals. Over time, a number of laboratories studying adult rodents have introduced transcardial perfusion with an ice-cold solution to decrease the animal’s body temperature, and therefore hypoxic damage to the brain, prior to decapitation. This procedure was successfully applied to produce cerebellar slices18, midbrain slices19, neocortical slices11,20, perirhinal cortex21, rat hippocampus10,22,23, olfactory bulb24, ventral striatum25, visual cortex26.
In spite of the advantages offered by transcardial perfusion and Na+ ion replacement in preparing slices from rat and in some brain regions in mice, mouse hippocampus remains one of the most challenging areas to protect from hypoxia13,20. To date, one of the most common approaches to slicing hippocampus from aging mice and mouse models of neurodegeneration involves the classical fast slicing of the isolated hippocampi27. In the protocol described here, we minimize the loss of ATP in the adult brain by introducing hypothermia prior to decapitation by transcardially perfusing the animal with ice-cold Na+– free NMDG-based artificial cerebrospinal fluid (NMDG-aCSF). Slices are then cut in ice-cold Na+-free NMDG-aCSF. With this enhanced protocol we obtain acute hippocampal slices from adult and aging mice that are healthy for up to 10 h after slicing and are appropriate for long-term field-recordings and patch-clamp studies.
The protocol is carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Stanford University Institutional Animal Care and Use Committee. Methods are also in accordance with the Policies of the Society for Neuroscience on the Use of Animals and Humans in Neuroscience Research.
NOTE: All mice were maintained in a pathogen-free environment. Wild-type mice on mixed C57Bl/ 6 x SV/ 129J genetic background were used here, unless otherwise noted.
1. Setup
2. Transcardial perfusion and brain extraction
3. Slicing
4. Recovery
We applied the above protocol to generate hippocampus slices from CamKIIa-Cre+;WT mice on a mixed genetic background C57Bl/ 6 x SV/ 129J, at P > 120. Large numbers of pyramidal cells in the CA1 field (Figure 2A) and subiculum (Figure 2B) appear in low contrast when observed under infrared differential contrast microscopy (IR-DIC), a hallmark of healthy cells in a slice preparation. With this preparation, a high rate of giga-ohm seals (>90%) is routinely achieved when targeting the healthiest cells approximately 20‒50 microns beneath the surface. For this success rate, it is important to use a high NA objective for IR-DIC, such as a 60x water-immersion objective, in order to achieve adequate visualization of pyramidal cells at this depth (Figure 2A,B).
Patch-clamp recordings from single neurons are easily achievable using this hippocampal slice preparation even in mice over six months of age. Figure 2C shows an example experiment using miniature excitatory postsynaptic currents (mEPSCs) recordings from CA1 neurons. Induction of chemical NMDA-LTD with bath application of 20 μM NMDA for 3 min lowers mEPSC frequency in CA1 cells when assessed 60 min post-NMDA treatment. This finding suggests that NMDA-LTD causes activity-dependent pruning of synapses in CA1 in older mice (results adapted from Djurisic et al.15). Change in mEPSC amplitude was not detected. During mEPSC recordings, CA1 cells were also filled with biocytin. Figure 2D reveals an intact dendritic arbor and healthy cell habitus of biocytin-filled CA1 pyramidal neurons. A robust distribution of the fluorescent dye throughout the cell allows for routine evaluation of dendritic spine properties under different experimental conditions.
Using field-recordings, long-term potentiation (LTP) of ~170% of CA3-CA1 synapses in slices from adult mice was readily observed, suggesting maintenance of signaling cascades needed for LTP (Figure 2E,F). Network connectivity needed for a robust field excitatory postsynaptic potential (fEPSP) signal is also preserved (Figure 2E). The ability to assess synaptic plasticity in hippocampal slices from adult or aging mice is especially relevant for mouse models of neurodegenerative diseases as their hallmark synaptic dysfunction develops later in life.
Together, our results demonstrate that an acute hippocampal preparation from adult and aging mice allows assessment of synaptic function at the level of both single cells and population of cells routinely, as long as transcardial perfusion and ice-cold NMDG-based solutions are used to minimize hypoxic damage.
Figure 1: Cutting method and recovery of transversal hippocampal slices from adult and aging mice. (A) A “60°” tool used for removing the 60° wedge from the rostral end of the brain, centered on the midline. (B) Positioning the hemispheres for cutting of transversal slices. An illustration of a 60° cut is shown on the left and the upper right panels; positioning of the two hemispheres on the surface with glue in manner shown in the lower right panel ensures a perpendicular orientation of dorsal hippocampus relative to the blade. This orientation results in transversal slices of hippocampus. Yellow structures are a 3D rendering of hippocampi within the rodent brain (gray). This illustration is adapted from SynapseWeb30. All the views are of the dorsal side of the brain. (C) Transversal hippocampal slices cut from a 4-month-old mouse in a recovery chamber. Please click here to view a larger version of this figure.
Figure 2: Patch-clamp and synaptic plasticity measurements in hippocampal slices from mice at P120‒180. (A) An example IR-DIC micrograph of CA1 region. (B) Example IR-DIC micrographs of subiculum. In both A and B, images are taken 3 h after cutting with a 60x water-immersion objective. Calibration bar is 10 µm. (C) The effect of chemical NMDA-LTD on mEPSCs in CA1 neurons from P120‒180 mice. Upper panel are example traces of mEPSCs recorded at baseline and after NMDA-LTD pulse. Lower left panel: NMDA-LTD resulted in lower mEPSC frequency. Lower right panel: mEPSC amplitude change after NMDA-LTD is not detected. N = 17 cells at baseline and n = 15 cells for NMDA-LTD, 6 mice. P = 0.004, t-test. This figure has been modified from Djurisic et al.15. (D) Example of biocytin-filled CA1 pyramidal cell. (E) Example of fEPSP signal from CA1 stratum radiatum after Schaffer collateral stimulation. The gray trace is obtained during baseline recording, and the black trace is observed 20 min after tetanic stimulation. Each trace is an average of 30 consecutive traces. (F) Cumulative average of long-term potentiation of CA3-CA1 synapses after 4 trains of 100 Hz induction; n = 23 slices from 8 WT mice at approximately P90. Please click here to view a larger version of this figure.
Artificial cerebrospinal fluid (aCSF) | ||||
Ingredients | Mw | Final conc. (mM) | g/2 Liters (10X) | g/Liter (1X) |
NaCl | 58.44 | 125 | 146.1 | – |
NaHCO3 | 84.01 | 26 | 43.68 | – |
KCl | 74.55 | 2.3 | 3.43 | – |
KH2PO4 | 136.1 | 1.26 | 3.44 | – |
Mg2SO4*7H2O | 203.3 | 1.3 | – | 1.3 ml of 1M stock |
CaCl2*2H2O | 147.02 | 2.5 | – | 2.5 ml of 1M stock |
Glucose*H2O | 180.2 | 25 | – | 4.5 g |
a) Add Mg2SO4*7H2O and CaCl2*2H2O from 1M stocks | ||||
b) aCSF 10X keep @RT | ||||
c) aCSF 1X @4°C for 24h | ||||
NMDG-aCSF | ||||
Ingredients | Mw | Final conc. (mM) | g/2 Liters (3X) | g/300 ml (1X) |
NMDG | 195.22 | 135 | 158.13 | – |
pH=7.4 with HCl | ||||
KCl | 74.55 | 1 | 0.4473 | – |
KH2PO4 | 136.1 | 1.2 | 0.9799 | – |
MgCl2*6H2O | 203.3 | 1.5 | 1.8297 | – |
CaCl2*2H2O | 147.02 | 0.5 | 0.4411 | – |
Choline Bicarbonate | 80% | 20 | – | 1238 µl |
Glucose*H2O | 180.2 | 10 | – | 0.54 |
a) Filter and store 3X stock solution at 4°C |
Table 1: Media formulations.
The protocol described here demonstrates that hippocampal slices obtained from adult and aging mice can remain healthy and viable for many hours after cutting. The slices prepared using this protocol are appropriate for patch-clamp recordings, as well as long-lasting field-recordings in the CA1 regions.
There are two critical steps in this protocol. First step is the transcardial perfusion step with an ice-cold solution. Fast clearance of blood is signaled by rapid change of liver color. Extracted brain should be off-white in color. If the brain remains pink, it means that systemic blood was not replaced with the cold NMDG-aCSF, and the drop in body temperature was not achieved. This could be caused by improper placement of the needle into the heart, or because the heart was punctured through. Brains from poorly perfused mice should not be used for slicing. The second critical step is the use of NMDG as a sodium ion substitute. A well-known earlier variation of the protocol that successfully uses sucrose as substitute for Na+ in rat brains10,31 does not produce sufficiently healthy hippocampal slices in mice (also see Ting et al.13). The use of NMDG as a sodium ion substitute is critical for mouse hippocampal slices.
While healthy hippocampal slices are reliably obtained using the described protocol, the hippocampal preparation from adult and aging animals remains challenging. Its difficulty also changes with different mouse lines and genetic backgrounds. Potential modifications to consider are additives to NMDG-aCSF and recovery-aCSF solutions like taurine, D-serine, or N-acetylcysteine (NAC), that could augment neuronal and synaptic function13,29, increase oxygenation29. Substitution of Cl- ions should also be considered during perfusion and cutting32. Use of an interface recovery chamber is another way to maintain increased oxygenation, which is especially relevant for long-term plasticity field-recordings. The described recovery chamber (Figure 1C) is modifiable for that purpose (e.g., a culture insert dish that is moistened from below by aSCF, could substitute the submerged mesh). Replacing steel blades with sapphire or ceramic blades could decrease tissue compression exacerbated by heavy myelination of white matter around hippocampus; this in turn could further improve the quality of neurons near the slice surface. Using microtomes designed to minimize the vertical vibration (e.g., zero-z in Leica VT1200S), is another modifiable step.
Other brain regions could be sliced using the protocol described here, with appropriate modifications in cutting angles. In addition, the stringency of the slice preparation can be adjusted, as different brain regions are sensitive to hypoxia to different degrees.. A protocol that uses an NMDG-aSCF has been reported for adult mouse neocortex and striatum slice preparations13,20; it uses an NMDG-aCSF that contains 30 mM NaHCO3 (i.e. it is not Na+-free)20, and in some instances transcardial perfusion is with NMDG-aCSF at room temperature13. However, for a region as susceptible to hypoxia as hippocampus, using Na+-free NMDG-aCSF and ice-cold transcardial perfusion could make a critical difference.
This protocol is applicable to the vast array of transgenic mouse lines modeling neurodegenerative diseases. Moreover, the protocol could be further modified to brain slices from other mammalian model species, as well. Together, this protocol could serve as a basis for a standardized preparation for acute hippocampal slices from aging animals, and thus facilitate comparisons across studies in the context of disease mechanisms.
The authors have nothing to disclose.
I thank Dr. Carla J. Shatz for advice and support, and Dr. Barbara K. Brott and Michelle K. Drews for critically reading the manuscript. The work is supported by NIH EY02858 and the Mathers Charitable Foundation grants to CJS.
“60 degree” tool | made in-house | ||
#10 scalpel blade | Bard-Parker (Aspen Surgical) | 371110 | |
1M CaCl2 | Fluka Analytical | 21114 | |
95%O2/5%CO2 | Praxiar or another local supplier | ||
Acepromazine maleate (AceproJect) | Henry Schein | 5700850 | |
Agar | Fisher | BP1423-500 | |
Beakers, measuring cylinders, reagent bottles | |||
Brushes size 00-2 | Ted Pella | Crafts stores are another source of soft brushes, with larger selection and better quality than Ted Pella. | |
CCD camera | Olympus | XM10 | |
Choline bicarbonate | Pfalz & Bauer | C21240 | |
Cyanoacrilate glue | Krazy glue | Singles | |
Decapitation scissors | FST | 14130-17 | |
Feather blades | Feather | FA-10 | |
Filter paper #2 | Whatman | Either rounds or pieces cut from a bigger sheet work well. | |
Forceps | A. Dumont & Fils | Inox 3c | |
Glass bubblers (Robu glass borosillicate microfilter candles) – porosity 3 | Robuglas.com | 18103 or 18113 | Glass bubblers are more expensive than bubbling stones used in aquaria. However, they are easy to clean and sterilize, and can last a long time. |
Glucose | Sigma-Aldrich | G8270 | |
HCl | Fisher | A144SI-212 | |
Ice buckets | |||
KCl | Sigma-Aldrich | P4504 | |
Ketamine HCl (KetaVed) | VEDCO | NDC 50989-996-06 | |
KH2PO4 | Sigma-Aldrich | P0662 | |
Leica Tissue slicer VT1000S | The cutting settings are 1 mm horizontal blade amplitude, frequency dial at 9, and speed setting at 2 | ||
Magnetic stirrers and stir bars | |||
Mg2SO4 x 7H2O | Sigma-Aldrich | 230391 | |
MgCl2 | Sigma-Aldrich | M9272 | |
MilliQ water machine | Millipore | Source for 18 Mohm water | |
Na-ascorbate | Sigma-Aldrich | A4035 | |
Na-pyruvate | Sigma-Aldrich | P8574 | |
NaCl | Sigma-Aldrich | S3014 | |
NaHCO3 | EMD | SX0320-1 | |
Needle 27G1/2 | |||
NMDG | Sigma-Aldrich | M2004 | |
Paper tape | |||
Peristaltic pump | Cole-Parmer | #7553-70 | |
Peristaltic pump head | Cole-Parmer | Masterflex #7518-00 | |
Personna blades | Personna double edge | Amazon | |
pH meter | |||
Recovery chamber | in-house made | ||
Scalpel blade handle size 3 | Bard-Parker (Aspen Surgical) | 371030 | |
Scissors angled blade | FST | 14081-09 | |
Single edge industrial razor blade #9 | VWR | 55411 | |
Spatulas | |||
Transfer pipettes | Samco Scientific | 225 | |
Upright microscope | Olympus BX51WI | ||
Xylazine HCl (XylaMed) | VetOne | 510650 |