This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.
Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 μm to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.
Electrophysiological measurement from living hippocampal slices in vitro is a powerful experimental approach with numerous advantages. The experimenter can use a microscope, micromanipulators, and a recording system to directly visualize and collect measurements from individual neurons in the tissue. Tissue slices are also very accessible to photostimulation or drug delivery for optogenetic, chemogenetic, or pharmacological experiments.
The hippocampal network generates highly synchronous population activity in vivo, visible as oscillations in the extracellular local field potential1,2,3,4,5. Brain slice methods have been leveraged to gain insight into the cellular and circuit mechanisms underlying these neuronal network oscillations. Foundational work from Maier et al. demonstrated that sharp wave-ripple complexes (SWRs) can emerge spontaneously in slices of the ventral hippocampus6,7. Subsequent studies from multiple investigators have gradually elucidated many aspects of SWRs, including the role of neuromodulators in regulating the network state of the hippocampus8,9,10 and the synaptic mechanisms that drive the in vitro reactivation of neuronal ensembles previously active during behavior in vivo11. Brain slice experiments have also provided insight into the gamma range oscillation (30–100 Hz), a distinct hippocampal network state believed to support memory encoding and recall12,13. Finally, recognizing the central role of the hippocampus and associated structures in the pathophysiology of temporal lobe epilepsy14,15, researchers have used hippocampal slice preparations to investigate the generation and propagation of epileptiform activity. Carter et al. demonstrated that combined hippocampal-entorhinal cortex slices prepared from chronically epileptic animals can spontaneously generate epileptiform discharges in vitro16. Subsequently, Karlócai et al. explored the mechanisms underlying epileptiform discharges in hippocampal slices by using modified artificial cerebrospinal fluid (ACSF) with altered ion concentrations (reduced Mg2+ or elevated K+) or added drugs (4AP or gabazine)17.
Investigators have developed numerous hippocampal slice approaches that differ in key ways: (1) the region of the hippocampus contained in the slice (dorsal, intermediate, or ventral); (2) the presence or absence of extrahippocampal tissues such as the entorhinal cortex; (3) the orientation used to cut slices (coronal, sagittal, horizontal, or oblique); and (4) the conditions under which the tissue is maintained after slicing (submerged fully in ACSF or held at the interface of ACSF and humidified, carbogen-rich air).
The choice of which slicing approach to use should be determined by the experimental objective. For example, transverse or coronal slices of the dorsal hippocampus maintained under submerged conditions have been used very effectively for the investigation of intrahippocampal circuitry and synaptic plasticity18,19,20. However, such preparations do not spontaneously generate network oscillations as readily as slices from the ventral hippocampus21,22,23. Although a state of persistent SWR activity can be induced by tetanic stimulation in transverse slices from the dorsal and ventral hippocampus24, spontaneous SWRs are more readily observed in ventral slices7,25.
An inherent physiological and anatomical distinction between the dorsal and ventral hippocampus is supported by studies performed both in vivo and in vitro26. Recordings in rats revealed strongly coherent theta rhythms throughout the dorsal and intermediate hippocampus, yet poor coherence between the ventral region and the rest of the hippocampus27. SWRs in vivo propagate readily between the dorsal and intermediate hippocampus, while SWRs that originate in the ventral hippocampus often remain local28. The associational projections originating from CA3 pyramidal neurons that reside in the dorsal and intermediate hippocampus project long distances along the longitudinal axis of the hippocampus. CA3 projections originating from ventral regions remain relatively local, and thus are less likely to be severed during the slicing process29,30. Ventral slices may, therefore, better preserve the recurrent network necessary to generate population synchrony. The propensity of ventral slices to generate spontaneous network activities in vitro may also reflect higher intrinsic excitability of pyramidal neurons or weaker GABAergic inhibition in the ventral hippocampus as compared to more dorsal regions31. Indeed, ventral hippocampal slices are more susceptible to epileptiform activity32,33. Thus, many studies of spontaneous physiological8,9,11,24 or pathological16,34,35,36 network oscillations have traditionally used a horizontal slicing approach, sometimes with a slight angle in the fronto-occipital direction, which yields tissue slices parallel to the transverse plane of the ventral hippocampus.
Network connectivity is unavoidably impacted by the slicing procedure as many cells in the slice will be severed. The angle and thickness of the slice and the tissue retained in the preparation should be considered to optimize connectivity in the circuits of interest. Many studies have utilized horizontal combined hippocampal-entorhinal cortex slices (HEC) to explore interactions between the two structures in the context of physiological or pathological network oscillations. Roth et al. performed dual recordings from the CA1 subfield of the hippocampus and layer V of the medial entorhinal cortex to demonstrate propagation of SWR activity through the HEC slice37. Many studies of epileptiform activity have used the HEC slice preparation to investigate how epileptiform discharges propagate through the corticohippocampal network16,35,36,38. It is important to note that preservation of the intact corticohippocampal loop is not a prerequisite for spontaneous SWRs, epileptiform discharges, or gamma oscillations; network oscillations can be generated in transverse slices of the dorsal or ventral hippocampus with no attached parahippocampal tissues21,22,23, 25,39,40,41. A more important factor for the spontaneous generation of network oscillations in hippocampal slices may be the thickness of each slice, as a thicker slice (400–550 μm) will preserve more connectivity in the CA2/CA3 recurrent network21,22,25.
Although angled horizontal HEC slices (cut with an approximately 12° angle in the fronto-occipital direction) have been used to study the functional connectivity of the corticohippocampal loop11,16,34,35,42, such angled preparations are not required for spontaneous network activity43,44,45. However, the use of an angled slicing plane does allow the investigator to selectively make slices that best preserve the transversely-oriented lamellae of either the ventral or intermediate hippocampus, depending on whether a downward or an upward angle is applied (Figure 1). This approach is conceptually similar to that used by Papatheodoropoulos et al., 2002, who dissected each hippocampus free and then used a tissue chopper to create transverse slices along the entire dorsal-ventral axis21. In the light of the aforementioned functional distinctions between the ventral and dorsal-intermediate hippocampus, investigators should consider the anatomical origin of slices when designing experiments or interpreting results. Using an agar ramp during the slicing procedure is a simple way to preferentially produce slices from either the intermediate or ventral hippocampus.
Hippocampal slices can be maintained in either a submerged chamber (with the tissue fully immersed in ACSF), or an interface-style chamber (e.g., Oslo or Haas chamber, with slices covered only by a thin film of flowing media). Interface maintenance enhances oxygenation of the tissue, which promotes neuronal survival and allows for sustained high levels of interneuronal activity. Traditionally, submerged recording conditions use a slower ACSF flow rate that does not provide adequate tissue oxygenation for stable expression of network-level oscillations. In submerged hippocampal slices carbachol-induced gamma oscillations are only observed transiently46,47, while they can be stably maintained in interface recording chambers10,48,49. As such, many studies of complex spontaneous activity in vitro have relied on interface recording chambers to investigate sharp-wave ripple complexes6,7,8,9,10,25,37, gamma oscillations10,13, and epileptiform activity16,38,45,47.
In a submerged-style recording chamber, an immersion microscope objective can be used to visualize individual cells and selectively target healthy-looking cells for recordings. The submerged preparation also allows fine control over the cellular milieu, as submersion facilitates rapid diffusion of drugs or other compounds to the tissue. Thus, a modified methodology in which stable network oscillations are maintained under submerged conditions represents a powerful experimental approach. This approach is exemplified by the work of Hájos et al., in which hippocampal slices recover in a simplified interface-style holding chamber for several hours before transfer to a modified submerged recording chamber with a high flow rate of ACSF (~6 mL/min) to enhance oxygen supply to the tissue12,48,49. Under these conditions, high levels of interneuron activity and stable spontaneous network oscillations can be maintained in a submerged recording chamber. This modified approach allows the investigators to perform visually guided whole-cell patch clamp recordings and characterize the contribution of morphologically identified cell types to carbachol-induced gamma oscillations12. SWRs can also occur spontaneously in submerged hippocampal slices with a fast flow rate of ACSF11,48,49. Maier et al. demonstrated that hippocampal slices that recovered in an interface chamber before transfer to a submerged recording chamber reliably exhibited spontaneous SWRs, whereas slices that recovered submerged in a beaker before transfer to a submerged recording chamber showed smaller evoked field responses, lower levels of spontaneous synaptic currents, and only very rarely exhibited spontaneous SWRs43. Schlingloff et al. used this improved methodology to demonstrate the role of parvalbumin-expressing basket cells in the generation of spontaneous SWRs44.
The following protocol presents a slicing method through which spontaneously active neurons in horizontal hippocampal slices can be recovered under interface conditions and subsequently maintained in a submerged recording chamber suitable for pharmacological or optogenetic manipulations and visually guided recordings.
All methods described here have been approved by the Institutional Animal Care and Use Committee at Columbia University (AC-AAAU9451).
1. Prepare solutions
2. Prepare agar ramp
3. Stage the slicing area
4. Transcardial perfusion
5. Extract the brain and cut slices
6. Perform local field potential (LFP) recordings of spontaneous activity
Presented here are representative recordings from HEC slices prepared as described in this protocol. Following recovery in an interface holding chamber (Figure 1C), slices are transferred individually to a submerged recording chamber (Figure 2B). The recording chamber is supplied with carbogen-saturated ACSF using a peristaltic pump (Figure 2A). The pump first draws ACSF from a holding beaker into a heated reservoir. Carbogen lines are placed into both the holding beaker and the heated reservoir to provide continuous oxygenation of the media. A pulsation dampener, consisting of a series of air-filled syringes, is positioned in between the peristaltic pump and the recording chamber to minimize the fluctuations in flow rate produced by rapid peristalsis. The air pocket in each syringe absorbs the changes in pressure caused by each cycle of the pump, so that the recording chamber receives a smooth and consistent flow of ACSF52,53. An inline heater positioned after the pulsation dampener ensures that the temperature of the ACSF is held at 32 °C as it enters the recording chamber.
In this example, the dual-surface superfusion recording chamber consists of three 3D-printed layers (Figure 2B). The bottom layer has a rectangular cutout to fit a coverslip, secured with vacuum grease. The middle layer contains the bottom half of an elongated oval chamber, with two horizontal supports. Nylon filament is strung across these supports (roughly every 0.5 mm) and secured with cyanoacrylate adhesive. The slice will rest on top of this strung filament. The top layer contains the upper half of the oval chamber along with small wells into which the silver chloride ground pellets can be placed. The elongated oval shape of the chamber is designed to promote fast laminar flow of ACSF.
Figure 3 presents representative recordings from HEC slices prepared according to this protocol. To initially assess slice health, field postsynaptic potentials (fPSPs) are evoked in the stratum radiatum (SR) using a pipette filled with 1 M of NaCl. In healthy slices, electrical stimulation should produce a fPSP with a small presynaptic fiber volley and a large postsynaptic potential with a rapid initial descent (Figure 3B, upper left). In healthy slices, spontaneous sharp-wave ripples (SWRs) are visible as positive deflections in the LFP in the stratum pyramidale (Figure 3B, lower left). In suboptimal slices evoked fPSPs show a large fiber volley and a relatively small postsynaptic potential, and such slices do not show spontaneous SWRs (Figure 3B, right). SWRs in vitro show characteristics consistent with published descriptions: a positive field potential in the SP layer with an overlaid high frequency oscillation, paired with a negative field potential in the SR layer (Figure 3C). A single SWR recorded in CA2 is indicated with an asterisk (Figure 3C, right). SWRs in HEC slices originate within CA2/CA3 recurrent circuits and propagate to CA1. A single SWR observed in the CA2 and CA1 SP layer is indicated with an asterisk (Figure 3D, right). In this representative example, the CA2 SWR (green) leads that in CA1 (blue) by several milliseconds, as shown in the overlay of the SWR envelope (filtered at 2–30 Hz) recorded in each region.
Figure 1: Preparation of horizontal angled hippocampal-entorhinal cortex (HEC) slices. (A)(i) After extracting the brain, perform two coronal cuts with a razor blade to remove the posterior and anterior portions of the brain. (ii) The agar ramp is formed of two angled portions glued to the microtome slicing platform. To prepare slices of the intermediate hippocampus, place the brain block onto the agar ramp with the anterior surface facing up the slope and making contact with the tall backing portion of the ramp. To prepare slices of more ventral hippocampus, place the brain block onto the agar ramp with the anterior surface facing down the slope, so that the posterior cut surface makes contact with the tall backing portion of the ramp. (iii) As each slice is freed, perform several more cuts with the scalpel to separate the hemispheres and remove unnecessary tissue. (B) Representative image of the resulting slice with cell nuclei labeled by DAPI. (C) In an interface recovery chamber, slices are placed on pieces of lens paper on top of a stainless steel or nylon mesh, level with the surface of the ACSF. A ceramic bubbler conveys carbogen into the chamber and a magnetic stir bar continually mixes the fluid in the chamber. A thin film of ACSF covers the top surface of the slice, enhancing diffusion of oxygen from the humid carbogen-rich air of the chamber. Please click here to view a larger version of this figure.
Figure 2: Dual surface superfusion recording chamber with pulsation dampener in the ACSF delivery tubing. (A) Diagram of the superfusion system. ACSF is warmed to 32 ˚C, constantly bubbled with carbogen gas, and delivered at approximately 8–10 mL/min using a peristaltic pump with a pulsation dampener consisting of a series of air-filled syringes. (B) The recording chamber consists of three 3D-printed layers, the middle of which is strung with nylon filament. The slice rests upon this strung filament and ACSF flows above and below the tissue. Please click here to view a larger version of this figure.
Figure 3: Representative recordings of spontaneous sharp-wave ripples from HEC slices. (A) A simplified diagram of the HEC slice showing the positions of the recording and stimulation electrodes. (B) Representative recordings of LFP activity from both an active, healthy slice and a suboptimal slice. The healthy slice (left, in green) shows large evoked field responses and spontaneous sharp-wave ripples (SWRs), visible as irregularly occurring positive deflections in the local field potential of the SP layer. In contrast, an unhealthy slice shows small evoked field responses and no spontaneous activity (right, in gray). (C) Representative recordings of SWRs in the CA2 region, consisting of a negative deflection in the LFP in the SR layer and a high frequency oscillation with an underlying positive deflection in the LFP in the SP layer. Peaks in each channel greater than three standard deviations of the signal amplitude are highlighted in red. A bandpass filter of 2–30 Hz isolates the underlying positive and negative envelope of the sharp wave in the SP and SR layer, respectively, while a bandpass filter of 80–250 Hz is used to isolate the high-frequency oscillation of the ripple in the SP layer. (D) SWRs in vitro propagate from CA2/CA3 to CA1. In these representative recordings, SWRs in CA2 (green, bottom) precede that in CA1 (blue, top) by several milliseconds. Peaks in each channel greater than three standard deviations of the signal amplitude are highlighted in red. Please click here to view a larger version of this figure.
molecular weight (grams / mol) | final concentration (mM) | grams / 1 L sucrose cutting solution | ||
sucrose | C12H22O11 | 342.3 | 195 | 66.749 |
sodium chloride | NaCl | 58.44 | 10 | 0.584 |
glucose | C6H12O6 | 180.08 | 10 | 1.801 |
sodium bicarbonate | NaHCO3 | 84.01 | 25 | 2.1 |
potassium chloride | KCl | 74.55 | 2.5 | 0.186 |
sodium phosphate monobasic anhydrous | NaH2PO4 | 137.99 | 1.25 | 0.173 |
sodium pyruvate | C3H3NaO3 | 110.04 | 2 | 0.22 |
stock concentration (M) | final concentration (mM) | milliliters / 1L sucrose cutting solution | ||
calcium chloride | CaCl2 | 1 | 0.5 | 0.5 |
magnesium chloride | MgCl2 | 1 | 7 | 7 |
Table 1: Composition of sucrose cutting solution. Begin with approximately 0.75 L of purified water that has been filtered to remove trace metals and other impurities. Dissolve each solid while mixing the solution with a magnetic stir bar. Once all solids are dissolved, bubble carbogen gas through the solution for 10 min. Add the MgCl2 and CaCl2 solutions and add water to bring the total volume to 1 L. Mix with a magnetic stir bar for 10 min to ensure the solution is uniformly mixed. The osmolarity should be between 315 and 325 mOsm, and the pH should be approximately 7.4.
molecular weight (grams / mol) | final concentration (mM) | grams / 2L ACSF | ||
sodium chloride | NaCl | 58.44 | 125 | 14.61 |
glucose | C6H12O6 | 180.08 | 12.5 | 4.502 |
sodium bicarbonate | NaHCO3 | 84.01 | 25 | 4.201 |
potassium chloride | KCl | 74.55 | 3.5 | 0.522 |
sodium phosphate monobasic anhydrous | NaH2PO4 | 137.99 | 1.25 | 0.345 |
ascorbic acid | C6H8O6 | 176.12 | 1 | 0.352 |
sodium pyruvate | C3H3NaO3 | 110.04 | 3 | 0.66 |
stock concentration (M) | final concentration (mM) | milliliters / 2L ACSF | ||
calcium chloride | CaCl2 | 1 | 1.6 | 3.2 |
magnesium chloride | MgCl2 | 1 | 1.2 | 2.4 |
Table 2: Composition of artificial cerebrospinal fluid. Begin with approximately 1.5 L of purified water that has been filtered to remove trace metals and other impurities. Dissolve each solid while mixing the solution with a magnetic stir bar. Once all solids are dissolved, bubble carbogen gas through the solution for 10 min. Add the MgCl2 and CaCl2 solutions and add water to bring the total volume to 2 L. Mix with a magnetic stir bar for 10 min to ensure the solution is uniformly mixed. The osmolarity should be between 315 and 325 mOsm, and the pH should be approximately 7.4.
There are several steps in this slicing protocol designed to promote tissue health and favor the emergence of spontaneous naturalistic network activity: the mouse is transcardially perfused with chilled sucrose cutting solution; horizontal-entorhinal cortex (HEC) slices are cut at a thickness of 450 μm from the intermediate or ventral hippocampus; slices recover at the interface of warmed ACSF and humidified, carbogen-rich air; during recordings slices are superfused with ACSF warmed to 32 °C and delivered at a fast flow rate with dual-surface superfusion in a submerged recording chamber.
Slice health is of paramount importance for the generation of network oscillations in vitro. Young animals will yield more healthy slices, and generally with juvenile or adolescent mice the transcardial perfusion step can be skipped. As animals age, it becomes increasingly difficult to make healthy slices, yet some investigations (such as disease models or longitudinal studies) necessitate the use of adult or aging animals. Dengler et al., for example, used transcardial perfusions in their preparation of HEC slices from pilocarpine-treated chronically epileptic adult mice50. With adult mice, it is beneficial to perform a transcardial perfusion with chilled sucrose cutting solution to cool the tissue, clear blood from the brain, and reduce metabolic activity before removing the brain from the skull51. It is important to note, however, that transcardial perfusions require training to be performed correctly and care must be taken to ensure that the procedure is carried out quickly and in a way that does not pose a risk to animal welfare. When possible, experiments should be designed to use young animals so as to preclude the use of transcardial perfusions. In a suboptimal slice preparation, there will not be enough healthy cells, particularly interneurons, to support ongoing network oscillations. To assess slice health during the experiment, it is useful to first record evoked field potentials (for example, with a stimulation pipette and a recording pipette in the stratum radiatum, SR). In a healthy slice, stimulation in the SR layer will evoke a large field postsynaptic potential (fPSP) with a relatively small presynaptic fiber volley (Figure 3).
The slices produced by this protocol are angled horizontal hippocampal-entorhinal cortex (HEC) slices. Importantly, neither the inclusion of parahippocampal tissue nor the angled cut are necessary for hippocampal slices to spontaneously generate network activities such as SWRs. Indeed, many studies have utilized horizontal or transverse hippocampal slices to interrogate aspects of physiological25,40,41,44 or pathological network oscillations33,38. In this protocol the placement of the brain onto an agar ramp allows the experimenter to selectively produce more slices from either the ventral or intermediate hippocampus (Figure 1), which may be beneficial for experimental objectives that take into account the functional heterogeneity that exists along the longitudinal axis of the hippocampus26,31. If the anatomical origin of the slices is not a factor, then the agar ramp can be excluded and a true horizontal cutting plane will yield slices of the intermediate-to-ventral hippocampus. As each horizontal tissue slice is freed, most extraneous parts of the slice can be removed with three simple cuts, leaving a roughly rectangular slice that contains the hippocampus and some surrounding tissue, including the parahippocampal region (Figure 1). Further dissection can be performed to remove all extrahippocampal tissues, but the inclusion of surrounding tissue is beneficial, in that the slice harp can be easily placed such that the nylon filaments do not rest across the hippocampus proper. As discussed above, the combined HEC slice is also a useful preparation with which to investigate a larger corticohippocampal network in the context of physiological37 or pathological16,35,36,38 network oscillations.
The key factor in this protocol is to optimize oxygen supply to the tissue, both during the recovery phase and during the recordings. Many studies of network oscillations are performed in slices that are transferred directly from the microtome to an interface recording chamber and allowed to recover with continual perfusion of fresh ACSF. After several hours of recovery, recordings can then be performed in the same interface chamber. Thus, slices are held at the interface of ACSF and humid carbogen-rich air for the full duration of the experiment. In the alternative methodology presented in this protocol, slices recover in an interface-style holding chamber for at least two hours before individual slices are transferred to a submerged-style recording chamber with sufficiently fast ACSF flow rates. Slices prepared under these conditions can exhibit stable gamma oscillations12 or spontaneous SWR activity43. Slice recovery in an interface-style holding chamber is a critical step: Maier et al. demonstrated that slices which recover in a beaker completely submerged in ACSF exhibit smaller evoked field potentials, less frequent spontaneous postsynaptic currents, and only rarely produce spontaneous network activity43. Similarly, Hájos et al. demonstrated that fast ACSF flow rates result in a higher frequency of spontaneous inhibitory postsynaptic currents, suggesting improved interneuronal activity49. During the recording period, dual-surface superfusion is not strictly necessary, provided the recording chamber holds a relatively small volume of ACSF delivered at a fast flow rate (at least 6 mL/min)43. The 3D printed recording chamber presented in this protocol (Figure 2B) is a relatively cost effective and a simple option, but there are also commercially available submerged recording chambers designed to hold smaller volumes, promote laminar flow of the media, or provide dual-surface superfusion (in contrast to circular recording chambers, for example, which allow excess dead volume of ACSF).
While this protocol allows one to record network oscillations without the requirement of a traditional interface recording chamber, there are several limitations. Although slices are held in the ACSF-air interface during the recovery period, they do not receive continual perfusion of fresh media as occurs in traditional Haas-style interface recording chambers. Slices must be transferred individually (using fine forceps) from the recovery chamber to the submerged recording chamber. Furthermore, fast flow rates can cause instability and motion artifacts problematic for some recordings, particularly if ACSF is delivered with a peristaltic pump. In order to maintain fast flow rates and minimize mechanical disturbances, a simple pulsation dampener can be integrated into the perfusion system (Figure 2). This pulsation dampener operates using the Windkessel effect52, in which empty syringes contain air pockets that act as elastic reservoirs, absorbing the fluctuating pressure generated by the rollers of the peristaltic pump53. However, incorporation of a pulse dampener can add length to the tubing that delivers ACSF to the recording chamber and impact oxygen supply to the slice. Flow rates should only be as fast as is necessary to yield stable network oscillations, and if a peristaltic pump is used it should ideally be a pump that uses a large number of rollers (> 12) to minimize the pulsation caused by peristalsis, preclude the use of a pulsation dampener, and ensure that the tubing that delivers ACSF to the recording chamber is as short as possible. Recordings performed with fast flow rates also necessitate large volumes of ACSF, which may be problematic if experiments require the addition of valuable or expensive drugs or compounds to the media. Although a dual-surface superfusion chamber requires a specially constructed recording chamber with two fluid inlets to deliver oxygenated media to both sides of the slice, spontaneous network oscillations can be observed with moderate ACSF flow rates48. This protocol utilizes both a fast flow rate (stabilized with a pulsation dampener) and a dual-surface superfusion chamber to improve the likelihood of observing spontaneous activity.
Finally, this protocol has a low yield with regards to the number of slices produced per animal. Horizontal slicing with a thickness of 450 μm yields a small number of HEC slices at the preferred orientation (in which the slice is effectively parallel to the transverse axis of the hippocampus). Of these slices, typically, only one or two per hippocampi exhibits spontaneous SWR activity, fewer than has been reported elsewhere43. Though thicker slices presumably contain a greater degree of recurrent connectivity, cutting slightly thinner slices (400 μm) may yield a greater number per mouse of transversely-oriented slices with SWR activity. In addition, the likelihood that multiple slices reliably exhibit spontaneous SWR activity may be higher in experiments that utilize true horizontal or downward angled slices of the ventral hippocampus. The current protocol incorporates the use of upward angled horizontal slices of the intermediate hippocampus, which may be less likely to exhibit spontaneous network activity in comparison to slices from the ventral hippocampus25,26,31. Finally, this protocol used slices from adolescent and adult mice. While transcardial perfusions can improve the quality of slices from older animals, the likelihood of observing spontaneous network activities may be improved by the use of slices from younger animals12,41,44.
In summary, this protocol presents a mouse brain slicing approach that yields angled horizontal hippocampal-entorhinal cortex slices from the intermediate or ventral hippocampal formation that can exhibit complex spontaneous network activity in the form of sharp wave-ripple complexes.
The authors have nothing to disclose.
The author would like to thank Steve Siegelbaum for support. Funding is provided by 5R01NS106983-02 as well as 1 F31 NS113466-01.
3D printer | Lulzbot | LulzBot TAZ 6 | |
Acute brain slice incubation holder | NIH 3D Print Exchange | 3DPX-001623 | Designed by ChiaMing Lee, available at https://3dprint.nih.gov/discover/3dpx-001623 |
Adenosine 5′-triphosphate magnesium salt | Sigma Aldrich | A9187-500MG | |
Ag-Cl ground pellets | Warner | 64-1309, (E205) | |
agar | Becton, Dickinson | 214530-500g | |
ascorbic acid | Alfa Aesar | 36237 | |
beaker (250 mL) | Kimax | 14000-250 | |
beaker (400 mL) | Kimax | 14000-400 | |
biocytin | Sigma Aldrich | B4261 | |
blender | Oster | BRLY07-B00-NP0 | |
Bonn scissors, small | becton, Dickinson | 14184-09 | |
borosilicate glass capillaries with filament (O.D. 1.5 mm, I.D. 0.86 mm, length 10 cm) | Sutter Instruments | BF150-86-10HP | Fire polished capillaries are preferable. |
calcium chloride solution (1 M) | G-Biosciences | R040 | |
camera | Olympus | OLY-150 | |
compressed carbogen gas (95% oxygen / 5% carbon dioxide) | Airgas | X02OX95C2003102 | |
compressed oxygen | Airgas | OX 200 | |
constant voltage isolated stimulator | Digitimer Ltd. | DS2A-Mk.II | |
coverslips (22×50 mm) | VWR | 16004-314 | |
cyanoacrylate adhesive | Krazy Glue | KG925 | Ideally use the brush-on form for precision |
data acquisition software | Axograph | N/A | Any equivalent software (e.g. pClamp) would work. |
Dell Precision T1500 Tower Workstation Desktop | Dell | N/A | Catalog number will depend on specific computer – any computer will work as long as it can run electrophysiology acquisition software. |
Digidata 1440A | Molecular Devices | 1-2950-0367 | |
digital timer | VWR | 62344-641 | 4-channel Traceable timer |
disposable absorbant pads | VWR | 56616-018 | |
dissector scissors | Fine Science Tools | 14082-09 | |
double-edge razor blades | Personna | BP9020 | |
dual automatic temperature controller | Warner Instrument Corporation | TC-344B | |
dual-surface or laminar-flow optimized recording chamber | N/A | N/A | The chamber presented in this protocol is custom made. A commercial equivalent would be the RC-27L from Warner Instruments. |
equipment rack | Automate Scientific | FR-EQ70" | A rack is not strictly necessary but useful for organizing electrophysiology |
Ethylene glycol-bis(2-aminoethyiether)- N,N,N',N'-teetraacetic acid (EGTA) | Sigma Aldrich | 324626-25GM | |
filter paper | Whatman | 1004 070 | |
fine scale | Mettler Toledo | XS204DR | |
Flaming/Brown micropipette puller | Sutter Instruments | P-97 | |
glass petri dish (100 x 15 mm) | Corning | 3160-101 | |
glucose | Fisher Scientific | D16-1 | |
Guanosine 5′-triphosphate sodium salt hydrate | Sigma Aldrich | G8877-250MG | |
ice buckets | Sigma Aldrich | BAM168072002-1EA | |
isoflurane vaporizer | General Anesthetic Services | Tec 3 | |
lab tape | Fisher Scientific | 15-901-10R | |
lens paper | Fisher Scientific | 11-996 | |
light source | Olympus | TH4-100 | |
magnesium chloride solution (1 M) | Quality Biological | 351-033-721EA | |
magnetic stir bars | Fisher Scientific | 14-513-56 | Catalog number will be dependent on the size of the stir bar. |
micromanipulator | Luigs & Neumann | SM-5 | |
micromanipulator (manual) | Scientifica | LBM-2000-00 | |
microscope | Olympus | BX51WI | |
microspatula | Fine Science Tools | 10089-11 | |
monitor | Dell | 2007FPb | |
MultiClamp 700B Microelectrode Amplifier | Molecular Devices | MULTICLAMP 700B | The MultiClamp 700B should include headstages, pipette holders, and a model cell. |
N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), (HEPES) | Sigma Aldrich | H3375-25G | |
needle (20 gauge, 1.5 in length) | Becton, Dickinson | 305176 | |
nylon filament | YLI Wonder Invisible Thread | 212-15-004 | size 0.004. This cat. # is from Amazon.com |
nylon mesh | Warner Instruments Corporation | 64-0198 | |
perstaltic pump | Harvard Apparatus | 70-2027 | |
Phosphocreatine di(tris) salt | Sigma Aldrich | P1937-1G | |
pipette holders | Molecular Devices | 1-HL-U | |
platinum wire | World Precision | PT0203 | |
polylactic acid (PLA) filament | Ultimaker | RAL 9010 | |
potassium chloride | Sigma Aldrich | P3911-500G | |
potassium gluconate | Sigma Aldrich | 1550001-200MG | |
potassium hydroxide | Sigma Aldrich | 60377-1KG | |
razor blades | VWR | 55411-050 | |
roller clamp | World Precision Instruments | 14041 | |
scale | Mettler Toledo | PM2000 | |
scalpel handle | Fine Science Tools | 10004-13 | |
slice harp | Warner | SHD-26GH/2 | |
sodium bicarbonate | Fisher Chemical | S233-500 | |
sodium chloride | Sigma Aldrich | S9888-1KG | |
sodium phosphate monobasic anhydrous | Fisher Chemical | S369-500 | |
sodium pyruvate | Fisher Chemical | BP356-100 | |
spatula | VWR | 82027-520 | |
spatula/spoon, large | VWR | 470149-442 | |
sterile scalpel blades | Feather | 72044-10 | |
stirrer / hot plate | Corning | 6795-220 | |
stopcock valves, 1-way | World Precision Instruments | 14054 | |
stopcock valves, 3-way | World Precision Instruments | 14036 | |
sucrose | Acros Organics | AC177142500 | |
support for swivel clamps | Fisher Scientific | 14-679Q | |
surgical scissors, sharp/blunt | Fine Science Tools | 14001-12 | |
syringe (1 mL) | Becton, Dickinson | 309659 | |
syringe (60 mL with Luer-Lok tip) | Becton, Dickinson | 309653 | |
three-pronged clamp | Fisher Scientific | 05-769-8Q | |
tissue forceps, large | Fine Science Tools | 11021-15 | |
tissue forceps, small | Fine Science Tools | 11023-10 | |
transfer pipettes | Fisher Scientific | 13-711-7M | |
tubing | Tygon | E-3603 | ID 1/16 inch, OD 3/16 inch |
tubing | Tygon | R-3603 | ID 1/8 inch, OD 1/4 inch |
vacuum grease | Dow Corning | 14-635-5D | |
vibrating blade microtome | Leica | VT 1200S | |
vibration-dampening table with faraday cage | Micro-G / TMC-ametek | 2536-516-4-30PE | |
volumetric flask (1 L) | Kimax | KIM-28014-1000 | |
volumetric flask (2 L) | PYREX | 65640-2000 | |
warm water bath | VWR | 1209 |