Oxygen consumption rates during three different neuronal activity states in the hippocampal CA3 network

Abstract

The brain is an organ with high metabolic rate. However, little is known about energy utilization during different activity states of neuronal networks. We addressed this issue in area CA3 of hippocampal slice cultures under well-defined recording conditions using a 20% O(2) gas mixture. We combined recordings of local field potential and interstitial partial oxygen pressure (pO(2)) during three different activity states, namely fast network oscillations in the gamma-frequency band (30 to 100 Hz), spontaneous network activity and absence of spiking (action potentials). Oxygen consumption rates were determined by pO(2) depth profiles with high spatial resolution and a mathematical model that considers convective transport, diffusion, and activity-dependent consumption of oxygen. We show that: (1) Relative oxygen consumption rate during cholinergic gamma oscillations was 2.2-fold and 5.3-fold higher compared with spontaneous activity and absence of spiking, respectively. (2) Gamma oscillations were associated with a similar large decrease in pO(2) as observed previously with a 95% O(2) gas mixture. (3) Sufficient oxygenation during fast network oscillations in vivo is ensured by the calculated critical radius of 30 to 40 μm around a capillary. We conclude that the structural and biophysical features of brain tissue permit variations in local oxygen consumption by a factor of about five.

Figure 1

Illustration of recording conditions and organotypic hippocampal slice cultures. (A) The custom-built acrylic glass unit contains two recording chambers that are used for maintenance of slice cultures on intact Biopore membrane inserts (left chamber, yellow asterisk) or acute brain slices (right chamber, white asterisk). (B) The recording solution (blue arrow) is supplied by an external peristaltic pump (not shown); the gas mixture (yellow arrows) is constantly exchanged. (C) The simplified scheme illustrates the functional design of the unit. The bottom part contains distilled water (mint rectangle) that is permanently warmed to 34±1°C. The gas mixture (20% O₂) is bubbled into the distilled water via a looped perforated plastic tube (white bar) for warming and humidification and guided (yellow arrows) to the intermediate floor. On the intermediate floor, the warmed and gas-saturated recording solution (blue bar) flows underneath the intact Biopore membrane insert (white rectangle with black lines; open from above) carrying slice cultures (purple bar). For recordings of local field potential (LFP) and partial oxygen pressure (pO₂), microelectrodes are positioned in slice cultures, through an opening in the cover plate and by using micromanipulators (not shown). (D) Morphologic characteristics of organotypic hippocampal slice cultures. The slice was maintained in culture for 12 days, then fixed, sectioned, and stained with cresyl violet. Note the ‘organotypic' preservation of hippocampal areas, CA1, CA3, and dentate gyrus (DG), including cell layers and functional connections (right image). Recordings of LFP and pO₂ depth profiles were conducted in stratum pyramidale in area CA3 (purple cell layer, left image). The scale bar denotes 500 μm.

Figure 2

Three different activity states of the hippocampal CA3 network. Recordings of the local field potential (LFP) were conducted in stratum pyramidale of area CA3 in organotypic hippocampal slice cultures. (A) Bath application of tetrodotoxin (TTX) (1 μmol/L) resulted in the complete absence of spiking after ∼20 minutes (TTX, n=8); compare with (B). (B) Spontaneous network activity (SPON, n=19) was present to a variable degree in all slice cultures investigated. (C) Bath application of cholinergic receptor agonist, acetylcholine (2 μmol/L) and acetylcholine-esterase inhibitor, physostigmine (400 nmol/L) resulted in persistent gamma oscillations after ∼30 minutes (GAM, n=8). The corresponding power spectra were calculated from data segments of 60 seconds (TTX, black; SPON, dark gray, GAM, light gray). Note the presence of gamma oscillations with a peak frequency of ∼50 Hz (lower power spectrum). A detailed analysis of cholinergically induced gamma oscillations in rat hippocampal slice cultures was published recently.

Figure 3

Depth profiles of partial oxygen pressure (pO₂) during the three different activity states. The activity states of the hippocampal CA3 network (Figure 2) were verified in each individual slice culture and pO₂ depth profiles were recorded with vertical steps of 16 μm. (A) Representative sample traces of pO₂ depth profiles in the absence of spiking (TTX, black trace), spontaneous network activity (SPON, dark gray trace), and cholinergically induced gamma oscillations (GAM, light gray trace). Tetrodotoxin (TTX, 1 μmol/L) as well as acetylcholine (2 μmol/L) and physostigmine (400 nmol/L) for induction of gamma oscillations were applied with the recording solution. (B) Quantification of lowest pO₂ values as determined during the three different activity states, i.e., TTX (n=8), SPON (n=19), and GAM (n=8). Lowest pO₂ values are indicated by black dots in the example traces (A). (C) Quantification of pO₂ values at five defined depths in slice cultures, i.e., at the upper slice surface (surface; depth level 1), in the middle of upper surface and slice core (depth level 2), in the slice core (core; depth level 3), in the middle of slice core and lower slice surface (depth level 4), and at the lower slice surface (bottom, depth level 5) for TTX (n=8), SPON (n=19), and GAM (n=8). Statistical significance is marked by asterisks (P<0.05).

Figure 4

Fittings of partial oxygen pressure (pO₂) depth profiles and calculation of oxygen consumption. Fittings (black lines) of representative pO₂ depth profiles during the three different activity states of the hippocampal CA3 network, i.e., tetrodotoxin (TTX) (A), spontaneous network activity (SPON) (B), and gamma oscillations (GAM) (C) as obtained from experiments illustrated in Figure 3. Note that pO₂ depth profiles show a convex behavior. The data were fitted from the upper slice surface to the lowest pO₂ value. (D) Quantification of oxygen consumption in the absence of spiking (TTX, n=8), during spontaneous network activity (SPON, n=19), and during gamma oscillations (GAM, n=8), given in arbitrary units (a.u.). Note that oxygen consumption during gamma oscillations was 2.2-fold and 5.3-fold higher as compared with spontaneous network activity and absence of spiking. Statistical significance is marked by asterisks (P<0.05).

Figure 5

Application of the model to estimate in vivo conditions. Experimentally determined values (small boxes) and simulated partial oxygen pressure (pO₂) depth profiles for the three different activity states, i.e., tetrodotoxin (TTX) (A), spontaneous network activity (SPON) (B), and gamma oscillations (GAM) (C), are shown by black lines (see also Figures 4A to 4C). In each panel, the first dark gray line illustrates the simulated pO₂ depth profile assuming that the convective component was absent and oxygen consumption was unaltered. When applying physiologic boundary conditions of 55 mm Hg (second dark gray line) or 28 mm Hg (third dark gray line) and again fixing the oxygen consumption to the same value, the resulting simulated pO₂ profiles reveal the radius around a vessel, in which neuronal tissue is sufficiently supplied with oxygen. In (C), the broken line depicts the pO₂ profile with the lowest oxygen boundary condition (∼40 mm Hg) that is still capable to fuel gamma oscillations at a distance of ∼35 μm midway between capillaries. Lower oxygen boundary conditions would require a hemodynamic response to maintain gamma oscillations.