There are currently no practical systems that allow extended regions (>5 mm2) of a tissue slice in vitro to be exposed, in isolation, to changes in ionic conditions or to pharmacological manipulation. Previous work has only achieved this at the expense of access to the tissue for recording electrodes. Here, we present a chamber that allows a tissue slice to be maintained in multiple solutions, at physiological temperatures, and preserves the ability to record from the slice. We demonstrate the effectiveness of the tissue bath with respect to minimizing the mixing of the solutions, maintaining the viability of the tissue, and preserving the ability to record from the slice simultaneously.
- tissue perfusion
- seizure generalization
the acute neural tissue slice is a valuable and widely used experimental preparation (Huang et al. 2012). Tissue, in vitro, is typically maintained in a chamber where it is perfused with an oxygenated solution (Croning and Haddad 1998). The ability to manipulate the tissue environment both focally and globally with changes to ionic conditions or pharmacological manipulation, in addition to electrical stimulation and electrophysiological recording, is a useful tool to elucidate tissue properties in health and disease (Scott et al. 2013). Fluid manipulation on these scales is the domain of microfluidics. The field of microfluidics potentially provides a powerful set of tools for such manipulations and is therefore currently being adopted by neuroscientists (Ahrar et al. 2013). A thorough review of microfluidic techniques applied to brain tissue slices is given in Huang et al. (2012). Recent examples of the application of microfluidics in neuroscience applications include focal pharmacological manipulation on the micrometer scale, less invasively than with conventional glass micropipettes (Mohammed et al. 2008; Queval et al. 2010; Tang et al. 2011), and improved perfusion of tissue slices allowing thicker tissue slices to be viably maintained (Choi et al. 2007; Hill and Greenfield 2011).
The global conditions of a tissue slice can be simply altered by changing the solution supplied to the tissue bath. The environment of regions of tissue on the scale of single cells can be modified using pressure ejection systems and micropipettes (Salierno et al. 2007; Smith and Cunningham 1983) as well as by microfluidic approaches (Queval et al. 2010). However, the ability to expose an extended region (>5 mm2) within a tissue slice to a change in conditions is limited. Work by Blake et al. (2007) used laminar flow to successfully maintain adjacent regions of tissue in different media. Their device did not, however, facilitate recording from the tissue surface. Access for recording electrodes was only possible along a single edge of the tissue. This was satisfactory for the medullary slices that were studied. However, for many applications, access for recording electrodes to the tissue surface is essential (Mody et al. 1987; Rutecki et al. 1985).
An example application for a chamber capable of exposing an extended region (>5 mm2) within a tissue slice to a change in conditions is in the study of the propagation of epileptiform activity across neural tissue. Epileptiform activity is commonly induced in neural tissue by the modification of the perfusing medium (Jefferys and Haas 1982; Konnerth et al. 1984). A chamber that perfuses regions of a tissue slice with different media would allow the study of the propagation of epileptiform activity, in the region treated with excitant solution, into an adjacent region, maintained in physiological medium. This provides a model for the study of the generalization of focal seizure activity.
Here, we present a tissue bath that uses laminar flow to maintain adjacent regions of tissue in different media while maintaining both the viability of the tissue and the ability to record from its surface. Such targeting has application in a wide range of network studies involving neural tissue and also has applications in the study of, for example, uterine and cardiac tissue. The chamber was manufactured using microstereolithography (MSL), a rapid direct digital manufacturing technique. This allowed bespoke chambers to be manufactured in <12 h.
MATERIALS AND METHODS
The requirements of the chamber were to:
expose extended regions (>5 mm2) within a tissue slice to different solutions, minimizing mixing at the interface between the solutions;
allow access to the tissue surface for recording;
perfuse both sides of the tissue to maintain its viability; and
maintain the tissue at a physiological temperature.
The chamber is composed of long, high-surface-area-to-volume-ratio, inflow channels before an accessible bath region (Fig. 1A). The channels were designed to minimize the Reynolds number of the perfusing medium flowing through them while allowing sufficient flow to maintain the viability of the tissue in the chamber. The purpose of the reduced Reynolds number flow is to eliminate turbulence before entry to the bath region. It was hypothesized (and tested, detailed below) that longer inflow channels would correspond to reduced mixing in the bath region. For practicality, the size of the bath was limited to the area of a standard microscope slide (75 × 25 mm). The length of the chamber channels was maximized given the overall size constraint and the size requirement of the bath region. This gave channels 37.2 mm long. The channels were 4 mm high and, for a two-channel chamber, 8.15 mm wide. For a two-channel chamber, water, at a flow rate of 5 ml/min, had a theoretical Reynolds number of 13.9 in the channels and 39.9 in the bath, both in a laminar flow regime (Brody et al. 1996).
A structure was developed to support the tissue, vertically, in the center of the flow, allowing perfusion across both surfaces. Oxygen reaches tissue in recording chambers by diffusion (Croning and Haddad 1998). Perfusion of the tissue across both surfaces increases the possible slice thickness that can be maintained hypoxia-free. Thicker slices provide a greater connected network for study. A similar component was designed to hold the tissue in place from above. Both the slice support and slice hold down were designed to fit in an accommodating slot in the bath region of the chamber (Fig. 1A). The horizontal bars connected the sides above the level of the fluid and, therefore, did not obstruct the flow and thereby introduce turbulence, which would increase mixing. The main structures of the support and hold down were manufactured using MSL. A 10-denier nylon mesh was stretched across the support, and 10-denier nylon fibers were attached across the hold down.
The insertion of microelectrodes into the chamber was expected to disrupt the laminar flow and cause mixing of the perfusing solutions. To counteract this effect, dividers were included on the slice support and hold down. These aligned with the divide between the channel inlets and thus reduced mixing in the chamber. The dimensions of the dividers were such that they did not come into contact with the tissue. The dividers reduced the perfusing flow to a small area of the tissue directly beneath them; they, however, tapered to 200 μm in width above the tissue surface to minimize this effect (Fig. 1, A and B). The maximum distance of any area of tissue from flowing perfusing medium in a 350-μm slice was 300 μm; diffusion could maintain tissue viability over this distance (Meme et al. 2009).
The bath region had a working area of 16.8 × 13.2 mm. This then tapered symmetrically toward a hypodermic needle connected to a pump for outflow. This avoided the eddy currents that would be expected in corners. Behind this was a region for an earth, allowing contact with the perfusing medium but not affecting the laminar flow.
The tissue bath was connected to inflow tubing with 7-mm long sections of 3-mm diameter capillary tubes. These were inserted into collars on the chamber.
The chamber consisted of a number of structures, manufactured by MSL, mounted on a standard microscope slide (75 × 25 mm). MSL is an additive layer manufacturing process that employs a photocurable resin to produce solid three-dimensional structures. Two-dimensional cross-sections are sequentially projected into a tray of liquid resin to cure regions where a solid is required and build up a three-dimensional structure.
The parts were designed in a computer-aided design package (SolidWorks 2011). The parts were then converted into a set of two-dimensional cross-sections at 25-μm intervals using EnvisionTEC (Gladbeck, Germany) RP. The parts were manufactured on a custom EnvisionTEC Perfactory Mini SLA system out of EnvisionTEC R11 (25-μm voxel depth) resin. After manufacture, the parts were washed with acetone and hardened in an EnvisionTEC Otoflash (1,000 flashes on each side; flash frequency: 10 Hz; spectral distribution: 300–700 nm; power: 200 W). This caused the polymerization of any unreacted resin on the surface of the parts. This was hypothesized (and tested, detailed below) to prevent contamination of the perfusing medium with resin from the chamber.
The components were mounted on a microscope slide, and 7-mm lengths of glass capillary were inserted into the chamber to form the interface for inflow tubes. These were secured with epoxy adhesive.
The nature of the manufacturing process meant that a chamber bespoke for an experiment could be manufactured rapidly. The build time for each component used here was 1 h and 42 min. Figure 1C shows the bath region of a chamber produced to target the entorhinal cortex region of a sagittal slice of cortex, used to investigate the origin of electrographic seizure activity. For such chambers, the inflow rate to each region had to be corrected to generate equal flow speeds for the different solutions to prevent shear stress between the solutions in the chamber.
Solutions were delivered (at the same flow rate) to the chamber through gas-impermeable Tygon tubing by a double-headed peristaltic pump (Sci-Q 400; Watson-Marlow, Cornwall, United Kingdom). A system of four valves allowed the solution supplied to each side to be changed without the introduction of air bubbles. Two syringes were connected to T-connectors immediately before the chamber. These were used to remove air bubbles from the channels in the chamber when initially priming.
Solutions were maintained at 34°C in a water bath to prevent degassing when heated before entry to the chamber.
To avoid the need for 2 heating systems (1 for each solution), a simple (and inexpensive) method of heating the 2 solutions was developed. A 125-ml glass beaker (positioned within the Faraday cage, close to the bath) containing water was maintained at 40°C. This was achieved by pumping (with a diaphragm pump, 0.35 l/min; RS, Northants, United Kingdom) water from a bath at 60°C, external to the Faraday cage, through a tube coiled in the beaker and back to the bath. The tubes containing the solutions were, at the end immediately before the bath, coiled and immersed in the water.
To test the temperature stability of the heating system, the temperature in the bath region of the device was measured for periods of 1 h (n = 3) at 10 Hz using a thermocouple connected to a TC-10 temperature controller (NPI Electronic Instruments, Tamm, Germany) and recorded using a CED Power1401 mk II digitizer controlled by Spike2 software (Cambridge Electronic Design, Cambridge, United Kingdom). This was compared with the temperature stability of solution in a commercially available bath (RC-27; Warner Instruments, Hamden, CT) heated using a commercially available system. The solution was heated by an HPT-2 heated perfusion tube (ALA Scientific Instruments, Harmingdale, NY), and the temperature was controlled by a TC-10 temperature controller (NPI Electronic Instruments). The temperature was recorded for the same periods using the same methods for both systems with a set temperature of 32°C. The mean temperature using the novel system (and the commercial system) was 31.57°C (29.46°C). The standard deviation was 0.18°C (0.12°C), and half of the maximum range was 0.47°C (0.37°C). The system we implemented was, therefore, deemed acceptable for use.
A tissue slice was transferred onto the mesh support, and the slice hold down was positioned on top to hold it in place (Fig. 1A). Slices were positioned relative to the divide between inflow channels as necessary for each experiment.
For the outflow, a hypodermic needle was held with its tip at the desired level of the bath. This was connected, through a second peristaltic pump (323; Watson-Marlow), to a waste container.
The effectiveness of the chamber in minimizing the mixing of two solutions was characterized using a fluorescent dye. The chamber was supplied with a 50 μM solution of fluorescein sodium salt (Sigma-Aldrich, Dorset, United Kingdom) in deionized water on one side and deionized water on the other. The chamber was uniformly illuminated with eight LEDs with peak emission at 458 nm (PLCC-4; RS) in an otherwise dark environment. An image of the chamber was captured with a VMS-004D microscope (Veho, Hampshire, United Kingdom). Images were corrected for intensity using a control region outside of the chamber. The fluorescence profiles at cross-sections 1, 6, and 12 mm into the chamber, along the direction of flow, were measured. Fluorescence was converted to concentration, and the interface width was measured, defined to be the distance over which the fluorescein concentration changed from 25 to 75% of its maximum.
The effectiveness of the channels in suppressing turbulent flow and therefore mixing was investigated. Four chambers were compared. These were manufactured with inflow channels of lengths uniformly spanning the possible range, giving channel lengths of 0, 12.4, 24.8, and 37.2 mm.
The effect on the width of the interface of the addition of microelectrodes and supporting net to the bath region was investigated for a chamber with full-length (37.2-mm) channels. The effect of the addition of net with divider, in addition to microelectrodes, tissue, and supporting net to chambers with no inflow channels (0 mm) and full-length inflow channels (37.2 mm), was also compared.
Fluorescence profiles were measured six times for each chamber and addition. The channel supplied with fluorescent solution was alternated and interleaved with controls during which both channels were supplied with the same solution.
The time for solution exchange in the chamber was measured using a fluorescent dye. The bath was illuminated and imaged as for experiments to measure mixing. The chamber was supplied on both sides with deionized water. This was then changed to a 50 μM solution of fluorescein sodium salt (Sigma-Aldrich) in deionized water. The fluorescence intensity was converted to concentration, and the time taken for the concentration of fluorescent solution to change from 5 to 95% of its maximum was measured. The solution exchange time was measured for a chamber with 4-mm high channels and a chamber with 2-mm high channels. For comparison, the exchange time in a commercial bath (RC-27; Warner Instruments) with solution depths of both 2 and 4 mm was also measured using the same method. In all cases, measurements were made (n = 3) both with the chambers empty and when containing a tissue slice and microelectrodes.
Water was collected after passing through the system and tested for contamination from the chamber. Deionized water was passed though the chamber at 5 ml/min, 31–32°C (as required of solutions for biological experiments), and collected. For comparison, deionized water was passed though the tubing of the system, with the chamber replaced with a petri dish, and collected. A mixture of <100 parts per million (ppm), by volume, R11 resin monomer, cross-linker, and associated solvents in deionized water was also prepared. One hundred microliters of R11 resin monomer, cross-linker, and associated solvents was mixed with 10 ml of deionized water for 30 min in a glass bottle on an orbital shaker. R11 resin is orange (because of the addition of photoinhibitors to aid layer thickness control), and the majority was seen to settle to the bottom of the bottle. One hundred microliters of the aqueous phase, that relevant to contamination of water-based solutions, was removed and mixed with 10 ml of deionized water. This resulted in a solution of the water-soluble components of R11 of ≪100 ppm. For each solution, a 2-ml aliquot was transferred to a 10-ml sample bottle with a crimp lid, fitted with a septum. The samples were heated to 80°C and exposed to a solid-phase microextraction (SPME) fiber (Supelco, Sigma-Aldrich) for 15 min. The SPME fiber was then inserted into a Scion SQ 451 gas chromatography-mass spectrometry (GCMS) system (Bruker, Bremen, Germany) and heated to 250°C. The GCMS column oven was heated from 50 to 280°C over 15 min. Mass spectra were recorded at 20 Hz over a mass range of 35–400 u.
Parasagittal slices of cerebellar vermis or cortex (350 μm) were prepared from male Wistar rats at postnatal days 18–21. All experiments were approved by the University of Warwick ethics committee. In accordance with the United Kingdom Animals (Scientific Procedures) Act 1986, rats were killed by cervical dislocation and decapitated. Either the cerebellum or cerebrum was rapidly removed. Parasagittal slices were cut on a Microm HM 650V microslicer (Carl Zeiss, Welwyn Garden City, United Kingdom) in cold (2–4°C) high-Mg2+, low-Ca2+ artificial cerebrospinal fluid (aCSF) composed of (in mM): 127 NaCl, 1.9 KCl, 8 MgCl2, 0.5 CaCl2, 1.2 KH2PO4, 26 NaHCO3, 10 d-glucose (pH 7.4 when bubbled with 95% O2-5% CO2). Slices were stored in experimental aCSF (1.0 mM MgCl2, 2.0 mM CaCl2) bubbled with 95% O2-5% CO2. The slices were maintained in a Gibbs chamber at 34°C for the 1st h after slicing and at room temperature for the remaining time before recording.
Individual tissue slices were transferred to the bath region of the chamber, maintained at 31–32°C, and continuously perfused at 5 ml/min with aCSF bubbled with 95% O2-5% CO2. Extracellular recordings were made with aCSF-filled electrodes, tip diameters 15 μm, placed on the surface of the tissue. The recordings were made using WPI ISO-DAM amplifiers (World Precision Instruments, Hertfordshire, United Kingdom) with band-pass filters 10 Hz to 3 kHz and digitized online (10 kHz) with a CED Power1401 mk II digitizer controlled by Spike2 software (Cambridge Electronic Design). Slices were maintained in the chamber for 20 min before the start of recording.
Schaffer collateral stimulation.
Schaffer collaterals were stimulated with a 50-μm diameter concentric bipolar stimulating electrode (FHC, Bowdoin, ME) from a Model 2100 Isolated Pulse Stimulator (A-M Systems, Sequim, WA). The resulting field excitatory postsynaptic potentials (fEPSPs) in CA1 were recorded to test slice viability in the chamber. Slices were maintained in the novel chamber for 3.5 h before stimulation. A paired-pulse protocol was implemented with a 50-ms interval between pulses, stimulating with 5 V for periods of 200 μs. The initial slope of the fEPSPs was measured, and the paired-pulse ratio was calculated. The evoked fEPSPs were compared with those from slices that were maintained in a Gibbs chamber for 3 h and then transferred to the chamber for a 30-min recovery period before stimulation. Slices that had been maintained in the two environments were interleaved. The initial slopes and the paired-pulse facilitation ratios of the evoked fEPSPs were compared with published results from other work (Manahan-Vaughan and Schwegler 2011; Sui et al. 2005).
Purkinje cells exhibit spontaneous action potentials, at a frequency of ∼50 Hz, in the absence of synaptic input (Raman and Bean 1999) and thus are active in in vitro cerebellar slices. This spontaneous activity was used as a functional measure of the effectiveness of the bath. Microelectrodes were placed on the surface of the Purkinje cell layer, one on each side of the chamber, such that spontaneous firing of Purkinje cells was recorded. Initially, both sides of the bath were perfused with aCSF (5 min). Following this, one side of the bath was perfused with the sodium-channel blocker TTX (1 μM; Ascent, Cambridge, United Kingdom) to terminate the spontaneous firing on that side of the bath. The other side was still perfused with aCSF. If the chamber functioned successfully, the spontaneous firing would be terminated on the side perfused with TTX but continue on the other side. After a further 5 min, 1 μM TTX was applied to both sides of the bath to confirm that firing on both sides could be abolished. In total, nine experiments were performed, and the side of the chamber to which TTX was first applied was alternated. For three of the experiments, the distance between the recording microelectrode on the side opposite to that initially supplied with TTX and the divide was minimized. This biologically tested the effectiveness of the solution separation. In all three cases, the microelectrode was <2 mm from the divide and in one case <1.5 mm from the divide.
Epileptiform activity recording.
Epileptiform activity was elicited in slices of cortex by perfusion with an excitant solution (experimental aCSF with 0 mM Mg2+, 5 mM K+; Mody et al. 1987). Slices were positioned such that approximately half of the cortex was on each side of the divide. The hippocampus did not cross the central divide of the bath. Microelectrodes were placed, one on each side of the bath, over layer five of the cortex. After transfer to the bath and placement of the microelectrodes, the slices were left to recover for 20 min before recording. Initially, both sides of the bath were perfused with aCSF for 5 min. Following this, one side of the bath was perfused with excitant solution, and the other with aCSF, for 30 min. If the chamber was functioning successfully, it was expected that this would elicit epileptiform activity on the side of the tissue exposed to excitant but not on the other side (perfused with aCSF). Subsequently, both sides of the bath were perfused with aCSF for 30 min to terminate any seizure activity. Both sides of the tissue were then perfused with excitant solution for 30 min. This was expected to elicit epileptiform activity on both sides of the tissue and thus confirm that they were viable and that the microelectrodes were correctly placed. Synchronicity of activity on both sides of the divide during this final period would demonstrate the viability of the region of the tissue under the divider.
The effectiveness of the inflow channels in reducing turbulence and mixing in the chamber was tested (as detailed above). The images of the bath region of the chamber with 0-mm channels (Fig. 2A) showed significant mixing arising from turbulent flow at the inlet. In contrast, images of the bath region of the chamber with 37.2-mm channels showed a straight, narrow interface (Fig. 2C). Profiles of cross-sections of the bath regions of the two chambers (Fig. 2, B and D, respectively) show a poorly defined interface in the case of the chamber with 0-mm channels and a well-defined, consistently positioned interface in the case of the chamber with 37.2-mm channels (the maximum length possible given the other design constraints). A >90% change in concentration occurred over <0.5 mm in the bath region of the full device. The interface width decreased with increasing channel length as did the variation in interface width (Fig. 2E). The interface width (25–75% change in concentration) was 3.7 ± 0.6 mm in the center of the bath region of the chamber with 0-mm channels and 0.4 ± 0.1 mm in the chamber with 37.2-mm channels (n = 6 for both). The visible effects of turbulent flow in the chamber with 0-mm channels, its apparent absence in the chamber with 37.2-mm channels, and the decrease in interface width with channel length all support the conclusion that the high-surface-area-to-volume-ratio inflow channels acted to suppress turbulent flow and, therefore, mixing.
The effect on the interface width of the introduction of microelectrodes and a supporting net to the chamber was measured. The presence of the microelectrodes or net in the images limited analysis to the profile at the cross-section at 12 mm. As expected, the addition of both microelectrodes and a supporting net caused an increase in interface width, to 2.7 ± 0.8 and 3.4 ± 0.6 mm, respectively (n = 6 for both; Fig. 2F). The replacement of the supporting net and hold down with ones with a divider effectively compensated for this effect. The interface width 12 mm into the bath region of a chamber with 37.2-mm channels, containing a support and hold down with dividers, a tissue slice and two microelectrodes, was 1.1 ± 0.2 mm. The dividers were, however, insufficient to prevent mixing independent of inflow channels. The interface width in a chamber with 0-mm channels, containing a support and hold down with dividers, a tissue slice and two microelectrodes, was 4 ± 0.8 mm.
After the change in solution supplied to the chamber, the fluorescence intensity increased uniformly across the bath region of the chamber (Fig. 3A). In contrast, there was turbulent mixing in the commercially available bath that resulted in stagnant regions, free from fluorescent solution, >20 s after the fluorescent solution reached the inflow of the bath (Fig. 3A). The increase in concentration of fluorescent solution in the bath region of the chamber increased sigmoidally (Fig. 3B) with a characteristic time scale less than or equal to that of the commercial bath under the same conditions (Fig. 3C). There was mixing of the two solutions in both the novel chamber and the commercially available bath. In the novel chamber, however, the mixing occurred in the inflow channels before the bath region. In both the novel chamber and the commercial bath, the exchange time was significantly longer with a solution depth of 4 mm than with a solution depth of 2 mm. When containing a tissue slice and two microelectrodes, the time for exchange with 4-mm deep solution for the novel chamber (and the commercial bath) was 68.9 ± 7.5 s (69.0 ± 2.9 s) and with a solution depth of 2 mm, 26.6 ± 2.3 s (36.0 ± 1.5 s).
Although mixing did occur between solutions supplied consecutively to the same side of the chamber, the time for exchange from 5 to 95% concentration was no worse than that for a commercially available bath. Turbulent mixing was isolated to the inflow channels before the region containing the tissue slice. This reduced areas of stagnant solution in contact with the slice.
There were no differences, above measurement noise, between the GCMS results from the solution that had passed through the chamber and the control solution that only passed through the associated tubing (Fig. 4A). The solution of R11 resin monomer, cross-linker, and associated solvents (≪100 ppm by volume) resulted in GC peaks consistently >50 times greater than the measurement noise. The upper limit on contamination was, therefore, 2 ppm by volume. Given the concentration of the R11 solution was an upper bound, the actual level of contamination was, however, much less than this. MSL, using R11 resin, provides a rapid technique for the productions of chambers bespoke for experiments. The resulting parts have been shown not to contaminate water-based solutions, at physiological temperatures, that pass through them. MSL using R11 resin could, therefore, be an effective manufacturing technique for many types of chamber with biological applications.
Schaffer Collateral Stimulation
The fEPSPs evoked in hippocampal slices maintained in the novel chamber were indistinguishable from those evoked in slices maintained in a Gibbs chamber (Fig. 4, B and C). The mean initial slope of the first fEPSP evoked in slices maintained in the novel chamber (and the Gibbs chamber) was −0.078 ± 0.012 mV/ms (−0.078 ± 0.014 mV/ms), and the paired-pulse facilitation ratio (for a 50-ms interval) was 2.14 ± 0.13 (2.15 ± 0.10). These results are consistent with published results from other work (Manahan-Vaughan and Schwegler 2011; Sui et al. 2005).
Slices maintained in the chamber for >3.5 h exhibited stimulus evoked responses indistinguishable from those evoked in slices maintained in standard chambers. There was, therefore, no evidence of any adverse effects either from contamination of the perfusing solution with components of the R11 resin (consistent with the results from tests with GCMS) or from a difference in perfusion.
The effectiveness of the chamber was tested using extracellular recordings from Purkinje cells. All slices (n = 9) exhibited spontaneous activity, demonstrating slice viability in the chamber. In all cases, application of 1 μM TTX to one side terminated the activity on that side (n > 3 for each side of the chamber) but had no effect on the opposite side, including those cases (n = 3) when the microelectrode on the side opposite to that initially perfused with TTX was <2 mm from the divide. The ongoing activity was shown to be sensitive to TTX with subsequent application to that side (Fig. 5). This demonstrated the effectiveness of the chamber in maintaining the solution separation under typical recording conditions with a slice present in the chamber.
Recording of Epileptiform Activity
The effectiveness of the chamber was further tested by eliciting epileptiform activity in slices of cortex. Extracellular recordings (n = 12) were made while implementing the perfusion protocol described above. This protocol was implemented with the side initially exposed to excitant being either the anatomically anterior region (n = 6) or the anatomically posterior region (n = 6). In all cases, epileptiform activity was recorded on the side perfused with excitant but not on the side perfused with experimental aCSF. Activity was recorded on both sides when both were perfused with excitant (Fig. 6). This demonstrated the effectiveness of the chamber in generating isolated activity in a tissue slice. As both anatomic regions were recorded as the first side perfused with excitant in isolation, the activity was not a result of the side exposed being more excitable.
The slices were alternated in orientation such that half of each set of six recordings was made with excitant first perfusing on each side of the chamber. This eliminated the possibility of an asymmetry in the perfusion.
In all cases, the epileptiform activity recorded when both sides were perfused with excitant was synchronized. Bursts of seizure activity, and the population spikes within them, were temporally correlated (Fig. 6C). This demonstrated the viability of the tissue between the central dividers. This was required for transmission and, therefore, correlation between the activity in the two regions of tissue. Synchronization was exhibited at the end of 95 min of recording. Including the time for microelectrode placement and that allowed for slice recovery, this demonstrated the chamber maintained the viability of entire slices for periods in excess of 2 h.
This form of protocol has application in the study of seizure propagation from pathological to physiological regions of tissue. This is exhibited in secondary generalization of seizure activity in vivo and is of significant research interest.
A chamber has been produced that allows extended regions of a tissue slice in vitro to be exposed, in isolation, to changes in conditions or drugs. The device facilitates the study of the propagation of epileptiform activity between regions of tissue in different ionic environments. The chamber could also be used to carry out experiments with a simultaneous control in coronal slices, with each side of a slice being maintained in different conditions. This would reduce bias as the regions being compared would be from the same position in the brain and when being tested would have been maintained for the same amount of time since dissection.
The effectiveness of the chamber has been shown to be preserved with the introduction of microelectrodes to the chamber to record from the slice. This simultaneous solution targeting and access for microelectrodes has not previously been achieved. The chamber was manufactured from R11 resin using MSL. This is an additive manufacturing technique allowing chambers bespoke for experiments to be produced rapidly. It has been shown that chambers manufactured from R11 using MSL do not contaminate water-based solutions that pass through them at physiological temperatures.
The chamber has been shown to maintain the viability of tissue slices for periods in excess of 3.5 h, perfusing them across all surfaces and maintaining them at a physiological temperature.
The design employed generalizes to varied geometries bespoke for experiments. In future work, chambers with greater numbers of channels will be developed.
This work was funded by the Engineering and Physical Sciences Research Council through the Molecular Organisation and Assembly in Cells (MOAC) Doctoral Training Centre.
No conflicts of interest, financial or otherwise, are declared by the authors.
M.G.T., J.A.C., and M.J.W. conception and design of research; M.G.T. and M.J.W. performed experiments; M.G.T. analyzed data; M.G.T. and M.J.W. interpreted results of experiments; M.G.T. prepared figures; M.G.T. drafted manuscript; M.G.T., J.A.C., and M.J.W. edited and revised manuscript; M.G.T., J.A.C., and M.J.W. approved final version of manuscript.
We thank Simon Leigh and Christopher Purssell for useful discussions and Eric Westenbrink for assistance with GCMS.
- Copyright © 2013 The American Physiological Society