This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.
The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer’s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.
The extensive exploration of the hippocampus started when Scoville and Milner reported the inability of a patient (H.M.) to form new, declarative memory after surgical removal of the hippocampus and nearby temporal lobe structures as a treatment for severe epilepsy1. From that moment on, the hippocampus has been studied extensively starting from general neuronal properties and functions up to the development of severe brain disorders, such as epilepsy and Alzheimer’s disease2,3,4,5. The hippocampus is part of the limbic system, consisting of a group of related brain structures involved in emotion and memory formation6,7. A dense network of several fiber pathways accomplishes a tight hippocampal connectivity to internal and external brain structures. These pathways include the medial and lateral perforant path (entorhinal cortex to dentate gyrus, CA3 – CA1 and subiculum)8, the mossy fiber path (dentate gyrus to CA3)9 and the Schaffer collateral/associational commissural pathway (CA3 to CA1)10 (Figure 1). The hippocampus presents one of the most broadly explored brain areas so far because of its highly conserved laminar organization of the neuronal layer formation, and the possibility to obtain vital neuronal cultures and brain slices with relative ease5.
Figure 1: Cartoon illustrating the different hippocampal regions and main fiber pathways. The different hippocampal regions are indicated by solid colored lines: entorhinal cortex (EC; black), dentate gyrus (DG; orange), Cornu Ammonis (CA) 3 (cyan), 2 (yellow), and 1 (magenta), and the subiculum (green). Fiber pathways are shown with a colored dotted line: the medial (MPP, red) and lateral perforant path (LPP, blue) (from the entorhinal cortex to the dentate gyrus, CA3, CA1, and subiculum), the mossy fiber pathway (MF, violet) (from the dentate gyrus to CA3) and the Schaffer collateral (SC, brown) (ipsilateral from CA3 to CA1)/associational commissural pathways (AC, light green) (contralateral from CA3 to CA1). Please click here to view a larger version of this figure.
Brain slice protocols often result in the loss of connections from more distant brain areas to the area of interest5. Moreover, the capillaries are no longer functional5 and the blood circulation is deprived11. Despite these limitations, brain slices are still primarily used for the investigation of neurophysiological functions of the hippocampus due to a number of advantages. First, the extraction of the hippocampus is fast12 and does not require many materials. The only essential instruments include a dissection kit, a laboratory water bath, access to carbogen and a vibrating microtome (vibratome)13. Other assets of the brain slice technique are the circumvention of the blood-brain-barrier (BBB) and the wash-out of endogenously released molecules before the start of the experiment5, which makes it possible to study the effect of drugs with relatively precise dosage control14. Furthermore, brain slices preserve the cyto-architecture and synaptic circuits within the hippocampus15,16, where the neuroanatomy and the local environment with neuronal connectivity and complex neuron-glia interactions are preserved4,11,17. Additionally, hippocampal fiber connections are predominantly unidirectional and hippocampal neurons have a high synaptic plasticity, which tremendously simplifies the collection and interpretation of high-quality electrophysiological recordings in order to understand neurological processes18,19. Importantly, brain slices present a valuable asset applicable in a wide range of different scientific techniques, spanning from molecular biological techniques over imaging recordings up to electrophysiological measurements12,20,21,22,23,24,25,26.
As outlined above, hippocampal brain slices present a powerful experimental tool to study structural and functional features of the synaptic connectivity. This offers the opportunity to assess the effects of chemicals or mutations on neuronal excitability and plasticity16.
Acute brain slice preparations are presenting a relatively sensitive technique and optimal slice quality is highly dependent on ideal experimental conditions, including the age of the animal, the method of euthanasia, the speed of dissection and slicing, the slicing solutions and parameters (e.g., slicing speed) as well as the conditions for slice recovery4. Therefore, a well-designed protocol is of uttermost importance and secures the reproducibility across different research units13.
Here, we provide a detailed protocol for acute horizontal hippocampal slice preparations, with the aim to preserve the integrity of the hippocampal lateral and medial perforant path and the mossy fiber pathway, allowing the investigation of dentate gyrus related processes9. We will describe in detail the key steps to dissect, extract, and horizontally slice the murine brain, followed by representative results of calcium-microfluorimetric recordings and field excitatory postsynaptic potential recordings (fEPSPs) under baseline conditions and during LTP induction protocols in brain slices of wild type C57BL/6J mice.
All animal experiments for this study were approved by the ethical review committee of the KU Leuven (Belgium) (P021/2012).
1. Preparation of high-sucrose slice solution and artificial cerebrospinal fluid (ACSF)
2. Preparation of the workspace for the brain dissection
3. Dissection and positioning of the murine brain
4. Horizontal slicing of the brain
5. Recovery of brain slices for electrophysiological recordings
6. fEPSP recordings in the medial perforant path (MPP) of the hippocampus
7. Calcium imaging recordings of brain slices
General overview of tools and critical steps needed for the protocol
Figure 2 presents all the necessary tools and critical steps for the preparation of horizontal acute hippocampal brain slices as described in this protocol. Generally, a limited number of key instruments are required, including a few dissection tools and a slice recovery chamber (Figure 2A), a laboratory water bath, and a vibratome (Figure 2B). Figure 2C–E visualize important steps and orientations of the brain and hemispheres during the slice preparation protocol. Figure 2F is an illustration of an expected result of horizontal brain slices.
fEPSP recordings in the medial perforant path
After the recovery period, the brain slices can be used for electrophysiological recordings of fEPSPs. Here, we used an upright microscope equipped with a multi-channel gravity-controlled perfusion system (Figure 3A and Figure 3B). A glass micropipette (~ 2 MΩ) was filled with ACSF solution and attached on top of a chloride-coated silver electrode that is mounted to an operational amplifier in circuit with a chlorinated bath electrode. fEPSPs were recorded and visualized with an amplifier and appropriate recording software by inserting the glass micropipette into the MPP of the hippocampus in the upper layer of the brain slice. fEPSPs were induced by stimulation with a 2-contact cluster microelectrode, applying different current intensities to the MPP upstream of the recording electrode. Note that this protocol is not intended to explain how to obtain MPP recordings, but simply uses recordings in the MPP as an example to demonstrate the success of the slice preparation protocol described here. If someone attempts to perform MPP recordings, certain controls (e.g., paired pulse recordings) might be necessary in order to ensure the proper recording site and distinguish the MPP from the LPP8,36,37.
Figure 3C illustrates a negative (left panel, low quality slice) and positive (right panel, high quality slice) example of an fEPSP recording. The negative example trace shows a large fiber volley (FV) amplitude that is even higher than the actual fEPSP amplitude (≈0.5 mV). In contrast, the high-quality slice example (right panel) shows a small FV to fEPSP ratio and a high fEPSP amplitude (>0.5 mV). The fiber volley is the signal that occurs upon depolarization of the stimulated neuronal fibers and therefore precedes the postsynaptic potentiation (fEPSP). The relation of FV to fEPSP properties provides important information about the preservation of the axonal and synaptic properties. High quality slices with intact nerve fibers should show a high fEPSP amplitude to FV ratio. On the contrary, low quality slices with impaired conduction properties will have a decreased fEPSP to FV ratio. Similarly, the viability of a brain slice can be analyzed by plotting fEPSP slopes versus the fiber volley amplitudes (Figure 3D).
Moreover, Input-Output curves (fEPSP slope and FV amplitude over stimulus intensity) are standardly used in order to determine the slice quality. Such curves are obtained by applying increasing current stimuli to the brain slice and by monitoring the subsequent fEPSP responses. Low quality brain slices show a reduced Input-Output curve due to suboptimal conduction properties of poorly preserved brain tissue (Figure 3E,F). Furthermore, Input-Output curves are necessary to define the ideal stimulation intensity range for the investigation of synaptic processes. Ideally, the stimulus intensity should be set around 50% of the intensity for maximal responses. At this chosen stimulus intensity, the fEPSP responses are highly sensitive for any changes in synaptic plasticity, which offers the opportunity to investigate both long-term potentiation (LTP) and long-term depression (LTD).
In order to study synaptic plasticity, the synaptic transmission of the brain slice (fEPSP slope) at the chosen 50% stimulus intensity is monitored for a longer time period (usually between 20–40 min) before the conditioning phase. Viable brain slices will have stable baselines, while brain slices with an unstable baseline cannot be used for further conditioning protocols in order to study synaptic plasticity of the brain circuits (Figure 3G, upper panel). fEPSP baseline recordings can also be useful in order to monitor drug effects on synaptic transmission itself (Figure 3G, lower panel). The mean of the recorded fEPSP baseline signals is typically used to normalize an fEPSP time course and is standardly set at 100%.
Synaptic plasticity can be studied by applying specific conditioning protocols to the brain slices. These protocols depend on the investigated brain circuit and the mechanism of interest (e.g., LTP or LTD). In order to induce LTP in the MPP of the dentate gyrus, a strong conditioning protocol is necessary due to the strong GABAergic inhibition that is present at the MPP synapses38. It is reported that the GABAergic inhibition is even more pronounced in brain slices prepared with high-sucrose slicing solutions39. Here, we use a protocol consisting of four stimulations of 1 s long 100 Hz pulses applied in a 5 min interval while being treated with the GABAA receptor antagonist Bicuculline (Figure 3H). The co-addition of NMDA and Bicuculline during the conditioning period results in an increased LTP (Figure 3H). Low quality of the slice and unstable synaptic transmission (fEPSP baseline) could result in altered or unsuccessful LTP and LTD induction. Therefore, it is of high importance to work with high quality slice preparations and use rigorous exclusion criteria (low fEPSP amplitude to fiber volley ratio (<3), small fEPSP slope (<0.5 mV/ms) or amplitude (<0.5 mV) and unstable fEPSP baseline (change of more than 5%) for unviable slices when investigating synaptic processes.
Calcium microfluorimetric measurements in the granule cell layer of the dentate gyrus
After recovery, a brain slice was incubated at room temperature with 2 µM of a calcium-sensitive dye for 1 h in carbogenated ASCF, shielded from light. The slice was transferred into a recording chamber (Figure 3A) on an upright fluorescence microscope equipped with a multichannel gravity-controlled perfusion system. Fluorescence emission images were acquired every 500 milliseconds after illumination at 488 nm (Figure 4A,B). Excitation was done with a Xenon lamp and a scanner mounted diffraction grating monochromator and image acquisition was performed with a computer-controlled CCD camera. During the measurements, the slice was treated with the NMDA receptor antagonist APV, which resulted in a decrease in the intracellular calcium concentration. Stimulation of the slice with an extracellular solution containing a high potassium concentration (50 mM) resulted in a massive influx of extracellular calcium due to the depolarization of the neurons and the opening of voltage-gated ion channels (Figure 4C).
Compound | Concentration (mM) | Molecular weight (g/mol) | Amount (g) |
KCl | 25 | 74.55 | 1.86 |
CaCl2 * 2H2O | 20 | 147.01 | 2.94 |
MgSO4 * 7H2O | 10 | 246.48 | 2.46 |
KH2PO4 | 12.5 | 136.08 | 1.7 |
Table 1: 10 x slice pre-solution (1 L).
Compound | Concentration (mM) | Molecular weight (g/mol) | Amount (g) |
NaCl | 125 | 58.44 | 7.3 |
KCl | 2.5 | 74.55 | 0.19 |
CaCl2 * 2H2O | 2 | from 1 M CaCl2 solution | 2 mL |
MgSO4 * 7H2O | 1 | from 1 M MgSO4 solution | 1 mL |
NaH2PO4 * 2H2O | 1.25 | 156.02 | 0.2 |
NaHCO3 | 26 | 84.01 | 2.18 |
Glucose * H2O | 25 | 198.17 | 4.95 |
Table 2: 1x ACSF (1 L) (osmolarity between 305–315 mOsm).
Compound | Concentration (mM) | Molecular weight (g/mol) | Amount (g) |
10x slice presolution | N/A | N/A | 25 mL |
Sucrose | 252 | 342.3 | 21.57 |
NaHCO3 | 26 | 84.01 | 0.55 |
Glucose * H2O | 10 | 198.17 | 0.49 |
Table 3: 1x high-sucrose slice solution (250 mL) (osmolarity between 320–325 mOsm).
Figure 2: Detailed information on the preparation of horizontal hippocampal brain slices. (A) Image of tools required for dissection and slicing of the rodent brain: (a) ±2 cm long and ±0.5 cm wide straps of filter paper (e.g., grade 413); (b) blade; (c) super glue; (d) pipette tip; (e) specimen plate (comes with vibratome); (f) 35 mm culture dish; (g) fine brush; (h) spatula; (i) curved forceps; (j) dissection scissors; (k) strong scissors (blade length above 10 cm); (l) plastic Pasteur pipette with wide opening (between 0.6 to 0.8 cm in diameter); (m) recovery chamber (self-made with 250 mL beaker, plastic ring, nylon mesh, piece of a 10 mL serological pipette); (n) 90 mm culture dish filled with ice and (o) square of filter paper on top of the chilled culture dish. (B) Picture of a vibratome with (a) holder of slice chamber filled with ice; (b) slice chamber; (c) carbogen line and (d) slicing razor blade. (C) Cartoon illustrating the orientation of the cut of the dorsal side of one hemisphere in order to prepare the brain for horizontal slicing (see step 3.9). (D) Isometric projection of the brain orientation on the specimen plate of the vibratome. (E) Cartoon illustrating a top view of the position of the two hemispheres on the specimen plate. (F) Cartoon showing the position of the hippocampus in a horizontal brain slice. The dentate gyrus (DG) and Cornu Ammonis (CA)–Subiculum (SB) regions of the hippocampus are indicated. (G) Picture of a recovery chamber with carbogenated ACSF containing ten freshly sliced horizontal brain slices. Please click here to view a larger version of this figure.
Figure 3: Electrophysiological recordings of hippocampal brain slices. (A) Image of a recording chamber with perfusion and suction, used under an upright microscope. A brain slice will be placed in the chamber and immobilized with a piece of a paper clip before the start of the recordings. (B) Bright field picture of a hippocampal brain slice under an upright microscope (10x objective). The dentate gyrus (DG) and CA3 region are indicated as well as the stimulation (bottom left) and recording (bottom right) electrodes, targeting the medial perforant path during fEPSP recordings. (C) Left: representation of a low-quality slice example of a fEPSP recording with a robust fiber volley and a small amplitude. Right: high quality slice example of a fEPSP recording. The gray line indicates the baseline level. The dotted lines point out the cut-off amplitude of 0.5 mV. (D) Plot of the fEPSP slope versus the FV amplitude for high quality (black; n=10) and low-quality brain slices (gray; n=4). Data represented as mean ± SEM. (E) Input-Output plot (fEPSP slope) for different stimulation intensities (µA) for high quality slices (black; n = 10) and low-quality slices (gray; n = 4)). (F) Same as in (E) but for the FV amplitudes versus the stimulus intensities. (G) Time course of three different baseline fEPSP recordings (slope of fEPSP in %; normalized to the mean fEPSP slope of the first 5 min). Upper panel represents a positive (black) and negative (red) example, where the latter has an unstable baseline due to omission of carbogen during the recording. Lower panel shows two stable baseline recordings in treated (after 20 min of stable baseline, AMPA receptors were blocked by application of the AMPA receptor antagonist DNQX (10 µM)) (blue) and untreated condition (black). (H) Time course of LTP recordings for different treatment conditions (indicated in lower panel). Black color for application of Bicuculline (20 µM) during conditioning and blue for co-application of Bicuculline (20 µM) and NMDA (10 µM) during conditioning. Arrows in upper panel indicate the time points where high frequency stimulation was applied (4 x 1s of 100Hz). Bar graph in lower panel represents the mean fEPSP slopes (%) for 50–60 min after LTP induction of the experiments shown in the upper panel (single representative recording for each condition). Please click here to view a larger version of this figure.
Figure 4: Calcium-microfluorimetry of hippocampal brain slices. (A and B) Fluorescence image (excited at 488 nm) (A) and corresponding heat map (B) of a horizontal hippocampal brain slice of the mouse brain. The dentate gyrus (DG), CA3 region, and an example of a region of interest (ROI) are indicated in panel A. (C) Time course of calcium responses (F488 nm) from a ROI in the dentate gyrus of an acute hippocampal brain slice during treatment with the NMDA receptor antagonist APV (50 µM) and solution containing high extracellular potassium (K+) (50 mM). The trace is normalized to the highest calcium response during high K+ perfusion and is baseline corrected for photobleaching. Please click here to view a larger version of this figure.
Although commonly used among the neuroscience community, brain slice preparations are also faced with several disadvantages. For instance, input and output connections to the brain areas of interest are no longer connected in a brain slice. Moreover, once isolated, the tissue starts degrading slowly over time and this process could alter the physiological conditions of the brain slice. This topic in particular is very concerning because most brain slice recordings are taking several minutes to hours, which results in long experimental days with recordings performed on tissue that was isolated up to 6–8 h before the start of the experiment. Furthermore, the cerebrospinal fluid and blood circulation get interrupted during slice preparations, which may lead to the lack of important endogenous compounds within a brain slice. And most obviously, the slicing procedure itself may cause mechanical tissue damage that might compromise the obtained results. However, the actual benefits of brain slice preparations are still outweighing their disadvantages, which is why they present a highly valued and employed technique in neuroscience research.
Acute hippocampal brain slices present a powerful and therefore widely used technique to investigate neuronal processes from a molecular level up to complex brain circuit studies. This is based on the ideal neuroanatomy of the hippocampus that can be easily preserved in a slice preparation18. Consequently, hippocampal brain slices are used in a wide variety of scientific research projects, including drug screenings17, studies of neuronal and synaptic properties involved in cognitive functions40,41, and investigations of pathological brain conditions14,42,43. However, a broad spectrum of different applications also causes a wide range of available slice preparation protocols that can differ in various parameters, such as dissection conditions and cutting plane orientation, among others. Therefore, the exact research question of a scientific project has to be determined in order to choose an appropriate slice preparation protocol.
The tissue chopper presents one of the oldest used techniques in order to prepare hippocampal brain slices44,45. The major advantages of this preparation method include the low cost of the chopper and the fast and easy usage46. However, tissue choppers cause mechanical stress that results in morphological alterations and cell death47. In comparison, the vibratome is a rather expensive machine and the time for slice preparation is significantly increased which might have an impact on the quality of the slice. However, the vibratome usually offers a more gentle manner of separating the slices from the tissue and allows to keep the brain nicely cooled and oxygenated over the entire isolation procedure, thereby improving slice properties46. Therefore, several groups are standardly employing this technique and have brought forward protocols for the preparation of acute hippocampal brain slices using the vibratome16,30,48. While some protocols provide only a few details for the slicing itself but rather focus on a specific application of such slice preparation48, others provide detailed slice protocols that differ in cutting plane or other protocol details (e.g., agarose embedding or slice/recovery solutions) given in this article27,30.
The protocol described here presents a straightforward method in order to prepare high quality acute horizontal hippocampal mouse brain slices from young animals. The protocol is particularly useful to preserve the perforant path (medial and lateral) that presents the hippocampal input pathway, which projects from the entorhinal cortex to the hippocampus8,49,50. Sagittal, coronal, as well as isolated hippocampus transverse slice preparations do not properly preserve the perforant path, which originates from mainly Layers II and V of the entorhinal cortex and projects to several areas within the hippocampus18. Due to the anatomical positioning of the entorhinal cortex in relation to the hippocampus, horizontal brain slices are a necessity in order to maintain fully intact perforant path fibers within the slice preparation31. Additionally, horizontal slicing ideally preserves the mossy fibers that project from the dentate gyrus to the CA3 neurons within the hippocampus9,30,50. Therefore, this preparation method is of high value for studies that investigate hippocampal input pathways and DG-related processes. In addition, this protocol allows the investigation of the Schaffer collateral pathway50. However, sagittal and coronal brain slice preparations are more commonly used when investigating CA3 to CA1 fiber projections, presumably because of their slightly faster preparation time that can increase the chance of obtaining high quality slices. Nevertheless, horizontal hippocampal slice preparations present a powerful research tool since it allows the preservation and investigation of all hippocampal fiber pathways within one slice hemisphere. This can be particularly useful when circuit responses are studied, for example, in multi electrode assay recordings.
A major concern when preparing brain slices is the proper preservation of the brain tissue. This is accomplished by several critical steps in our protocol, including a fast dissection, the continuous and sufficient oxygenation and cooling of the tissue, and the protection of the brain tissue by use of the protective cutting method with a low-sodium, high-sucrose slicing solution39,51. Despite the fact that the protocol described here yields a success rate around 90%, potentially additional protective steps might be required when working with tissue derived from older or genetically diverse animals or when trying to preserve a specific cell population. Several methods were already reported to protect sensitive brain tissue preparations. These methods include the use of NMDG-based slicing solutions to reduce the sodium permeation52, the use of high magnesium levels in the slicing solution in order to block NMDA receptor activity53, and the prolonged use of protective solutions also during the recovery period23. All of these measures will result in a reduced excitotoxicity. Additionally, a trans-cardial perfusion with ice-cold protective ACSF solutions is often employed and necessary when working with older animals27.
Acute hippocampal brain slices are ideally suited and extensively used for electrophysiological studies for reasons such as the high amplitude signals that can be obtained from a relatively thick (300–500 µm) acute brain slice, which guarantees a high signal to noise ratio11. Standardly used electrophysiological applications include extracellular field recordings and intracellular whole-cell recordings in a voltage- or current-clamp mode. In order to acquire high quality electrophysiological data, the slice health is of primary concern and can be guaranteed by strictly following the presented protocol. However, as slice preparations present a highly sensitive technique, a quality check should be routinely included before the start of each experiment. Several parameters can be used as quality check of the slice and are standardly assessed via Input-Output curves and baseline fEPSP or EPSC recordings19. Nevertheless, it should be noted that suboptimal electrophysiological properties can arise from experimental errors such as electrode positioning, orientation or even damage and do not solely represent the health of the prepared slice. Therefore, it is advisable to perform additional quality controls such as simple visualization and assessment of the cells under a 40x objective or a DAPI nucleus staining. Such quality checks can be used to confirm constant slice health over several slice preparation sessions.
Calcium microfluorimetry presents a less commonly used technique to study hippocampal brain slices. However, this technique is of additional value to the standard extracellular and intracellular electrode recordings, as it allows to visualize and quantify intracellular calcium fluxes, which are of high importance in neuronal and synaptic signaling. Changes in intracellular calcium concentrations are involved in neurotransmitter vesicle release, postsynaptic potential generation, regulation of synaptic plasticity and axonal nerve conduction54,55,56. As an illustration of this technique (Figure 4), we made use of a commercially available calcium dye. Inarguably, treatment of tissue slices with calcium dyes can yield difficulties such as an increased experimental time frame as well as inefficient loading of lower situated neuronal cells. However, variations on this technique could be used to circumvent these technical challenges. For instance, it is possible to combine calcium measurements and patch clamp recordings in hippocampal slices. In this way, a calcium fluorescent dye could be loaded into a specific cell through the patch pipette, allowing the measurements of calcium dynamics in one specific cell of interest57. Alternatively, genetically engineered animals expressing the calcium indicator, GCaMP58, either in the whole brain, or driven by a cell-specific promotor, could be used. Interestingly, brain tissue from GCaMP animals with a direct linker to a protein of interest could provide opportunities to determine the neuronal expression pattern or investigate the involvement in calcium sparks and waves.
Altogether, we provide the guidelines for the successful preparation of healthy and viable horizontal hippocampal brain slices from mice for electrophysiological and imaging recordings. This methodology is very useful to access neurological changes that occur in brain pathologies that are described in the dentate gyrus.
The authors have nothing to disclose.
We thank the Electrophysiology unit of the VIB-KU Leuven Center for Brain and Disease Research under the supervision of Dr. Keimpe Wierda and Prof. Dr. Joris De Wit for the use of their research facilities. Furthermore, we thank all the members of the Laboratory of Ion Channel Research and the Laboratory of Endometrium, Endometriosis and Reproductive Medicine at the KU Leuven for their helpful discussions and comments.
This project has received funding from the Research Foundation-Flanders (G.084515N and G.0B1819N to J.V.) and the Research Council of the KU Leuven (C1-funding C14/18/106 to J.V.). K.P. is a FWO [PEGASUS]2 Marie Skłodowska-Curie Fellow and received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (665501) with the Research Foundation Flanders (FWO) (12T0317N). K.H. is a Postdoctoral Fellow of the Research Foundation Flanders, Belgium (12U7918N).
Anesthesia chamber | home made – Generic | N/A | plexiglas |
Anesthesia vaporizer | Dräger & MSS International Ltd | Isoflurane Vapor 19.3 & MSS Isoflurane | to vaporize isoflurane for rodent anesthetization |
Barrels for the perfusion system | TERUMO | Hypodermic syringes without needle | https://www.terumotmp.com/products/hypodermics/terumo-hypodermic-syringes-without-needle.html |
Bicuculline methiodide | hellobio | HB0893 | https://www.hellobio.com/bicuculline-methiodide.html |
Borosilcate glass capillaries | Science Products | GB150F-8P | https://science-products.com/en/shop/micropipette-fabrication-1/capillary-glass-for-micropipette-pullers/borosilicate-glass-capillaries/borosilicate-filament-polished |
Calcium chlorid dihydrate | Merck | 102382 | https://www.merckmillipore.com/BE/en/product/Calcium-chloride-dihydrate,MDA_CHEM-102382?ReferrerURL=https%3A%2F%2Fwww.google.com%2F |
Calcium Imaging software | Till Photonics | LiveAcquisition v2.3.0.18 | |
Carbogen tank | Air Liquide | Alphagaz mix B50 | Gasmixture CO2/O2: 5/95, purity 5 |
Cluster microelectrode | FHC | CE2C55 | https://www.fh-co.com/product/cluster-microelectrodes/ |
Culture dish (35 mm) | Corning Life Sciences | 353001 | https://ecatalog.corning.com/life-sciences/b2c/US/en/Cell-Culture/Cell-Culture-Vessels/Dishes%2C-Culture/Falcon®-Cell-Culture-Dishes/p/353001 |
Culture dish (90 mm) | Thermo Fisher Scientific | 101VR20 | https://www.thermofisher.com/order/catalog/product/101R20#/101R20 |
Curved forceps | Fine Science tools | 11270-20 | https://www.finescience.de/de-DE/Products/Forceps-Hemostats/Dumont-Forceps/Dumont-7b-Forceps/11270-20 |
D-AP5 | hellobio | HB0225 | https://www.hellobio.com/dap5.html |
D-(+)-Glucose monohydrate | Sigma Aldrich | 16301 | https://www.sigmaaldrich.com/catalog/product/sial/16301?lang=en®ion=BE |
Digital CMOS camera | HAMAMATSU | ORCA-spark C11440-36U | https://www.hamamatsu.com/eu/en/product/type/C11440-36U/index.html |
Dissection scissors | Fine Science tools | 14058-09 | https://www.finescience.de/de-DE/Products/Scissors/Standard-Scissors/Fine-Scissors-ToughCut®/14058-09 |
DNQX | hellobio | HB0262 | https://www.hellobio.com/dnqx-disodium-salt.html |
EMCCD camera | Andor | iXon TM + DU-897E-CSO-#BV | https://andor.oxinst.com/products/ixon-emccd-cameras?gclid=CjwKCAjw97P5BRBQEiwAGflV6ULsKjXfhN2YZxtvsWAmF4QghyXZKuqYHVMa6KU9JyS80ATQkSKeBBoCIM0QAvD_BwE |
EPC10 USB Double Patch Clamp Amplifier | HEKA Elektronik | 895278 | https://www.heka.com/sales/brochures_down/bro_epc10usb.pdf |
Filter paper | VWR | 516-0818 | grade 413 |
Fine brush | Raphael Kaerell | 8204 | Size #1 |
18G needle | Henke Sass Wolf Fine-Ject | 18G X 1 1/2" 4710012040 | https://www.henkesasswolf.de/cms/de/veterinaer_produkte/produkte_vet/einmalkanuelen/hsw_henke_ject_einmalkanuelen/ |
Isoflurane | Dechra Veterinary Products | Iso-Vet 1000mg/g | 250 ml bottle |
Loctite 406 | Henkel Adhesive technologies | Loctite 406 | Super glue |
Magnesium sulfate heptahydrate | Merck | 105886 | https://www.merckmillipore.com/BE/en/product/Magnesium-sulfate-heptahydrate,MDA_CHEM-105886?ReferrerURL=https%3A%2F%2Fwww.google.com%2F |
Micromanipulator | Luigs & Neumann | SM-10 with SM-7 remote control system | https://www.luigs-neumann.org |
Microscope (for calcium imaging) | Olympus | BX51WI | https://www.olympus-lifescience.com/de/microscopes/upright/bx61wi/ |
Microscope (for ephys recordings) | Zeiss | Axio Examiner.A1 | https://www.micro-shop.zeiss.com/de/de/system/axio+examiner-axio+examiner.a1-aufrechte+mikroskope/10185/ |
Microscope light source | CAIRN Research | dual OptoLed power supply | https://www.cairn-research.co.uk/product/optoled/ |
Monochromator | Till Photonics | Polychrome V | |
N-Methyl-D-aspartic acid (NMDA) | Sigma Aldrich | M3262 | https://www.sigmaaldrich.com/catalog/product/sigma/m3262?lang=en®ion=BE |
Oregon Green® 488 BAPTA-1 | Invitrogen Molecular Probes | #06807 | 10x50ug |
Osmometer | Wescor | 5500 vapor pressure osmometer | to verify osmolarity of salt solutions |
Peristaltic pump | Thermo Fisher Scientific | Masterflex C/L 77120-62 | https://www.fishersci.be/shop/products/masterflex-peristaltic-c-l-dual-channel-pump-2/p-8004229 |
pH meter | WTW | inoLab series pH 720 | https://www.geminibv.nl/wp-content/uploads/manuals/wtw-720-ph-meter/wtw-inolab-ph-720-manual-eng.pdf |
Pipette puller | Sutter Instrument | P-1000 | https://www.sutter.com/MICROPIPETTE/p-1000.html |
Potassium chlorid | Chem-lab | CL00.1133 | https://www.chem-lab.be/#/en-gb/prod/1393528 |
Potassium dihydrogen phosphate | Merck | 104873 | https://www.merckmillipore.com/BE/en/product/Potassium-dihydrogen-phosphate,MDA_CHEM-104873?ReferrerURL=https%3A%2F%2Fwww.google.com%2F |
Razor blade to prepare hemispheres | SPI supplies | Safety Cartridge Dispenser – Pkg/10 | GEM Scientific Single Edge Razor Blades |
Razor blade for vibratome | Ted Pella Inc | 121-6 | double edge breakable style razor blades (PTFE-coated stainless steel) |
Recovery chamber | home made – Generic | N/A | to collect and store brain slices in (see details in manuscript) |
Scissors | Any company | N/A | Blade should be well sharpened and at least 15 cm long for easy decapitation |
Silver electrode wire | Any company | for recording and reference electrodes | |
Sodium dihydrogen phosphate dihydrate | Merck | 106342 | https://www.merckmillipore.com/BE/en/product/Sodium-dihydrogen-phosphate-dihydrate,MDA_CHEM-106342?ReferrerURL=https%3A%2F%2Fwww.google.com%2F |
Sodium hydrogen carbonate | Alfa Aesar | 14707 | https://www.alfa.com/en/catalog/014707/ |
Sodium chlorid | Fisher Scientific | S/3160/60 | https://www.fishersci.co.uk/shop/products/sodium-chloride-certified-ar-analysis-meets-analytical-specification-ph-eur/10428420 |
Software for field recordings | HEKA Elektronik | PatchMaster | https://www.heka.com/downloads/software/manual/m_patchmaster.pdf |
Spatula | Sigma Aldrich | S9147-12EA | https://www.sigmaaldrich.com/catalog/product/sigma/s9147?lang=en®ion=BE |
Stimulator | A.M.P.I | ISO-FLEX | http://www.ampi.co.il/isoflex.html |
Sucrose | VWR International Ltd. | 102745C | https://es.vwr-cmd.com/ex/downloads/magazine/lupc_userguide_uk.pdf |
Tubing for carbogen, perfusion and suction lines 1 | Warner Instruments | 64-0167 | Tygon tubing (TY-50) for standard valve systems |
Tubing for carbogen, perfusion and suction lines 2 | Fisher Scientific | 800/100/200 & 800/100/280 | Smiths Medical Portex Fine Bore LDPE Tubing |
Vacuum pump | home made – Generic | N/A | |
8 valve multi-barrel perfusion system | home made | N/A | consists of barrels, tubing and a self-made automated valve control (specifications of all purchased parts can be found in this Table) |
Magnetic valves (to control the perfusion lines) | NResearch Inc. | p/n 161P011 | https://nresearch.com/ |
Vibratome | Leica | 14912000001 | Semi-automatic vibrating blade microomei VT1200 |
Water bath | Memmert | WNB 7 | https://www.memmert.be/wp-content/uploads/2019/09/Memmert-Waterbath-WNB-7.en_.pdf |
Water purification system | Merck | Synergy millipore | to obtain highly purified water |
12-well plates | Greiner Bio-One | CELLSTAR, 665180 | http://www.greinerbioone.com/UserFiles/File/Catalogue%202010_11/UK/3680_005-Kapitel1_UK.pdf |