Organotypic hippocampal slice cultures are typically cultured on porous membrane inserts (Stoppini et al., 1991
) or roller-tubes (Gahwiler, 1981
; Gahwiler et al., 1997
). Initially, we tested whether postnatal day 7 (P7) rat organotypic hippocampal slices cultured on 30 mm diameter porous membrane inserts (Millipore, Billerica, MA) for 14 days in vitro
could be transferred into XF Islet Capture Microplates for measurements of O2
consumption. Islet microplates consist of a modified XF24 V7 plate (Seahorse Bioscience) containing a 3 mm diameter 1 mm deep microchamber at the bottom of wells. An accompanying fitted nylon mesh insert (70 μm pore size) is designed to trap pancreatic islet tissue () but can be adapted for other tissue types. The XF24 lowers a retractable probe with a spotted O2
sensor into each well, sealing off a small volume of medium above the inserts in which tissue O2
consumption is assessed (Gerencser et al., 2009
; Wu et al., 2007
). When rat hippocampal slices were detached from porous membrane inserts and transferred to XF Islet Capture Microplates ~1 hour prior to measurements as free-floating tissue beneath capture screens, O2
consumption rates were noisy and did not respond to the ATP synthase inhibitor oligomycin or the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP, data not shown).
Fig. 1 Schematic diagram of hippocampal slices attached to nylon mesh inserts (“Capture Screens”) within XF Islet Capture Microplates. A retractable probe limits O2 diffusion to the tissue when in the measurement position, enabling estimation (more ...)
To overcome the problems of free floating tissue moving laterally and vertically with respect to the O2 sensitive fluorophore spots and the poor drug responsiveness, P7-8 rat or mouse organotypic hippocampal slices were cultured directly on the XF nylon mesh inserts before transfer to the XF measurement plates (, ). Slices cultured for up to 18 DIV on the inserts were generally viable, with the majority of slices exhibiting low to moderate staining by propidium iodide (PI), a nuclear dye excluded by healthy cells. In a few slices, PI staining localized strongly to the CA1 pyramidal layer, indicating extensive cell death within this region. Images in illustrate the range of PI staining observed in slices, with representative of the majority.
Representative brightfield (A) and propidium iodide (PI)-stained fluorescent images (B-D) of organotypic mouse hippocampal slices cultured on nylon mesh inserts for 12-18 DIV. The same slice is depicted in A and B. Scale bars, 50 μm.
Next, we tested whether stable O2 consumption rates (OCR) and drug responses could be measured from rat organotypic slices cultured directly on the nylon inserts and then inverted into XF Islet Capture Microplates. For these pilot experiments we used an assay medium consisting of a relatively standard artificial cerebrospinal fluid solution (see Materials and Methods) that was supplemented with glucose (25 mM) and pyruvate (0.23 mM). We observed a large variability in OCR from 14 DIV slices of a single rat brain, with a mean ± SD of 703 ± 307 pmol O2/min (n=12 slices). In the absence of drug additions, respiration exhibited a slow, progressive decline but remained at 72% of the initial baseline after two hours (, filled black circles). Addition of the uncoupler FCCP (1 μM) caused an initial stimulation of OCR (, filled gray circles). However, further additions lead to a progressive decrease in OCR, most likely due to toxicity, with OCR reaching ~50% of the baseline rate at ~1.5 hours after the first addition. Titration with the ATP synthase inhibitor oligomycin led to a slow drop in OCR to ~30% of the initial rate, with a peak response at ~1 hour subsequent to the first addition (, open circles). The majority (~90%) of hippocampal O2 consumption was attenuated by the mitochondrial complex III inhibitor antimycin A (10 μM), indicating that it was of mitochondrial origin.
Fig. 3 Oligomycin and FCCP titrations for organotypic rat hippocampal slices. Absolute (A) and baseline-normalized (B) O2 consumption rates (OCR) of 14 DIV organotypic rat hippocampal slices exposed to successive additions of respiratory modulators (arrows) (more ...)
Because organotypic slice cultures are typically restricted to brain tissue from immature animals while neurodegenerative disorders are most prevalent in the aging population, we tested whether the same technique could be applied to acute rat hippocampal slices. Acute slices were attached to the nylon inserts within two hours of slicing by centering them on a small volume of chicken plasma and initiating the formation of an O2 permeable clot by thrombin addition (see Materials and Methods). The basal OCR of acute rat slices of 798 ± 377 pmol O2/min (n=10 slices) did not differ significantly from that of organotypic rat hippocampal slices cultured for 14 DIV. As in organotypic slices, FCCP caused a slight (~13%) stimulation of OCR while oligomycin decreased OCR to ~53% of baseline after 1 hour (). Of 10 slices, one slice displayed an unstable baseline (170% drop in OCR from the first to the third measurement) and residual OCR that was antimycin A-insensitive. Antimycin A-insensitive (non-mitochondrial) O2 consumption was greater than 30% of the total OCR for two additional slices. These slices had a reduced ratio of mitochondrial to non-mitochondrial O2 consumption compared to the majority of the slices, likely due to an increased number of cells lacking functional mitochondria. Therefore they were considered damaged and were excluded from analysis here () and in subsequent experiments.
Fig. 4 Oligomycin and FCCP titrations for acute rat hippocampal slices. Baseline-normalized O2 consumption rates (OCR) of acute rat hippocampal slices exposed to successive additions of respiratory modulators (arrows) are shown without (A) and with (B) exclusion (more ...)
One of the technical challenges of adapting XF24 technology for use with hippocampal slices is preventing tissue hypoxia. Organotypic hippocampal slice cultures are adapted to culture at atmospheric (20-21%) O2
while acute hippocampal slices are typically maintained in aCSF continuously bubbled with 95% O2
. Because O2
diffusion is limited within tissue, hyperoxic conditions of 20% or 95% O2
(with respect to normal tissue pO2
) are intended to prevent hypoxia in the slice core and are particularly important with thicker sections. While O2
tension at the surface of organotypic cultures 180-210 μm thick was previously measured at ~148 mm Hg in air (near the expected atmospheric value), O2
tension in the slice core was measured at ~51 mm Hg (Huchzermeyer et al., 2008
). This pO2 approaches measured pO2
values of rat cortex, which vary with energy demand and cortical layer between ~15-40 mm Hg (~2-5% O2
) (Erecinska and Silver, 2001
; Grote et al., 1996
; Liu et al., 1995
). Although pO2
in the slice core is tolerable with respect to physiological normoxia, O2
levels in the XF24 drop during the measurement period when the microchamber above the slices is sealed off from the atmosphere. Thus, pO2
in the slice core may transiently decline below physiologically normoxic levels if tissue O2
consumption rates are sufficiently high.
While it was not possible to get a measure of pO2 in the slice core using the XF24, the measurement of O2 levels in the aCSF are expected to approximate pO2 in the surface layer of cells. We found that even under basal conditions (i.e. in the absence of uncoupler to stimulate respiration), the pO2 in aCSF dropped below 80 mm Hg (, open circles) in a slice respiring with an initial rate of 1029 pmol O2/min. The pO2 remained above 125 mm Hg (, filled circles) in a sister slice respiring at 329 pmol O2/min. However, the basal OCR of this slice was more than a standard deviation below the average OCR (703 ± 307 pmol O2/min). These data suggest that the majority of rat slices likely experience transient hypoxia in the slice core by the end of each two minute measurement period, even in the absence of respiratory stimulation.
Fig. 5 Medium O2 levels (mm Hg) during measurements of O2 consumption by rat (A) and mouse (B) organotypic slices. In A, representative fast and slow respiring rat slices are depicted (open circles and filled circles, respectively, see text). Arrows represent (more ...)
Because transient hypoxia during measurements may influence the physiology of the slice, we tested organotypic mouse hippocampal slices which contain fewer cells per slice compared to rat slices of the same thickness. Under basal conditions, O2 remained above 140 mm Hg () for representative fast and slow O2 consuming slices that had a basal OCR of 250 pmol O2/min (open circles) and 118 pmol O2/min (filled circles), respectively. Furthermore, O2 remained above 125 mm Hg for the fast respiring slice even after OCR was stimulated to 405 pmol O2/min by the uncoupler FCCP and excess exogenous substrate (10 mM pyruvate). Therefore, mouse organotypic hippocampal slices were used in all subsequent experiments to further optimize hippocampal respirometry.
In respirometry experiments with neuronal cells and isolated synaptic nerve terminals (synaptosomes), bovine serum albumin (BSA) was used as a surrogate for extracellular protein (Choi et al., 2009
; Jekabsons and Nicholls, 2006
; Yadava and Nicholls, 2007
). Treatment of mouse organotypic hippocampal slices with 1 μM FCCP in the absence of BSA led to little initial OCR stimulation. However, there was a delayed peak response that was similar but less variable in the presence of BSA (4 mg/ml, ). In the absence of BSA, increasing the FCCP concentration to 5 μM abolished detection of respiratory stimulation () and led to progressive inhibition (data not shown), likely due to toxicity (see Fig. and ). In contrast, with BSA present, peak respiratory stimulation was observed at the initial measurement after 5 μM FCCP addition (). Glucose (15 mM) was used as the energy substrate in these experiments and pyruvate (10 mM) was co-injected with FCCP to exclude the possibility that substrate supply was rate-limiting for uncoupled respiration. Additional injections of FCCP up to a cumulative total of 15 μM failed to further stimulate OCR in either the absence or presence of BSA (data not shown). Because BSA facilitated the determination of maximal OCR, it was included in subsequent experiments.
Fig. 6 The effect of BSA on FCCP-stimulated O2 consumption rates (OCR). Baseline-normalized initial and peak OCR of organotypic mouse hippocampal slices exposed to FCCP (1 or 5 μM) in the absence (n=4) or presence (n=3) of BSA (4 mg/ml). Initial OCR (more ...)
Next, we sequentially injected oligomycin, FCCP, pyruvate, and antimycin A to estimate coupling efficiency, maximal respiration in the presence of glucose, maximal respiration in the presence of exogenous complex I-linked substrate, and non-mitochondrial OCR, respectively (). As in rat hippocampal slices, antimycin A strongly inhibited organotypic mouse slice O2 consumption. Inhibition of the ATP synthase by oligomycin attenuated mitochondrial (antimycin A-sensitive) OCR by >75%, demonstrating that the majority of O2 consumption was coupled to ATP synthesis. FCCP addition restored OCR to ~basal values while the addition of pyruvate was necessary to elevate uncoupled OCR above the endogenous basal rate. Baseline OCR exhibited high variability (), as was the case with rat hippocampal slices. Normalizing OCR to the third basal rate prior to drug additions () considerably reduced variability.
Fig. 7 Estimation of coupling efficiency, maximal respiratory capacity, and the effect of exogenous pyruvate. Absolute (A) and baseline-normalized (B) O2 consumption rates (OCR) of 17 DIV organotypic mouse hippocampal slices exposed to successive additions of (more ...)
Finally, to provide a preliminary indication of how many replicates would be required to detect differences in bioenergetic parameters following an experimental treatment, we evaluated the variability in OCR of 10 total slices derived from three different wild type mice of the same litter that were expected to display similar responses to the sequential addition of oligomycin, FCCP plus pyruvate, and antimycin A (). When comparing absolute OCR, there was high variability among the mice in basal rates and drug responses (). The variability in responses was greatly reduced when O2 consumption rates for each slice were normalized to the basal rate (). This finding suggests that most of the variability was due to the technical variability of measurements (i.e. number of cells per slice, proximity of slices to the O2 sensor, etc., see discussion) rather than biological variability of bioenergetic parameters. We performed a power analysis to assess how many slices per animal would be required to detect a 20% difference between hypothetical control and experimental groups, assuming equal group size and groups of 5 or 10 animals (Table I). A power of 0.8 was readily achievable for comparisons of normalized data at a significance level of 0.05 but required a large number of slices for comparisons of absolute OCR. Overall, the data in in combination with the power analysis illustrate that reproducible normalized O2 consumption data can be obtained from organotypic hippocampal slices while cautioning against comparisons of absolute OCR without a high number of replicates.
Fig. 8 Assessment of inter-animal variability. Absolute (A) and baseline-normalized (B) O2 consumption rates (OCR) of 14-15 DIV organotypic mouse hippocampal slices exposed to successive additions of respiratory modulators (arrows) are shown for three different (more ...)