Relationship between oxygen consumption and neuronal activity in a defined neural circuit

Abstract

Background: Neuronal computations related to sensory and motor activity along with the maintenance of spike discharge, synaptic transmission, and associated housekeeping are energetically demanding. The most efficient metabolic process to provide large amounts of energy equivalents is oxidative phosphorylation and thus dependent on O₂ consumption. Therefore, O₂ levels in the brain are a critical parameter that influences neuronal function. Measurements of O₂ consumption have been used to estimate the cost of neuronal activity; however, exploring these metabolic relationships in vivo and under defined experimental conditions has been limited by technical challenges.

Results: We used isolated preparations of Xenopus laevis tadpoles to perform a quantitative analysis of O₂ levels in the brain under in vivo-like conditions. We measured O₂ concentrations in the hindbrain in relation to the spike discharge of the superior oblique eye muscle-innervating trochlear nerve as proxy for central nervous activity. In air-saturated bath Ringer solution, O₂ levels in the fourth ventricle and adjacent, functionally intact hindbrain were close to zero. Inhibition of mitochondrial activity with potassium cyanide or fixation of the tissue with ethanol raised the ventricular O₂ concentration to bath levels, indicating that the brain tissue consumed the available O₂. Gradually increasing oxygenation of the Ringer solution caused a concurrent increase of ventricular O₂ concentrations. Blocking spike discharge with the local anesthetics tricaine methanesulfonate diminished the O₂ consumption by ~ 50%, illustrating the substantial O₂ amount related to neuronal activity. In contrast, episodes of spontaneous trochlear nerve spike bursts were accompanied by transient increases of the O₂ consumption with parameters that correlated with burst magnitude and duration.

Conclusions: Controlled experimental manipulations of both the O₂ level as well as the neuronal activity under in vivo-like conditions allowed to quantitatively relate spike discharge magnitudes in a particular neuronal circuitry with the O₂ consumption in this area. Moreover, the possibility to distinctly manipulate various functional parameters will yield more insight in the coupling between metabolic and neuronal activity. Thus, apart from providing quantitative empiric evidence for the link between physiologically relevant spontaneous spike discharge in the brain and O₂-dependent metabolism, isolated amphibian preparations are promising model systems to further dissociate the O₂ dynamics in relation to neuronal computations.

Keywords: Extraocular motoneurons; Oxidative phosphorylation; Ventricle; Xenopus laevis.

Fig. 1.

Measurements of O₂ levels in isolated preparations. a, b Photomicrograph, depicting an isolated head of a stage 53 tadpole (a₁), a schematic transverse section of the hindbrain (a₂), and a cross-sectioned head (b) at a rostro-caudal level indicated by the trapezoid in a₁; red and blue circles and arrows in a₂ indicate movements of the O₂ electrode within the grid (white dots in b), used to construct the O₂ profile (b). c Dual recordings of O₂ concentrations in the bath and above the floor of the IV^th ventricle in steps of 0.2 mm. d O₂-concentration profile (mean ± SEM) of a midline depth track above the IV^th ventricle (a₂) in control (intact; color-coded) and metabolically inactive (EtOH-fixated; gray) preparations. e, f Recording of the ventricular O₂ concentration in an EtOH-fixated preparation (e) during temporary increase of the bath O₂ level to 650 μmol/l (gray area), and of an intact preparation (f) after bath-application of KCN (light pink area; 500 μmol/l); note that KCN causes an adjustment of the ventricular O₂ level to the bath O₂ level (single arrow in f) that remains matched (double arrow in f) when the bath O₂ level is further increased (dark pink area). g Boxplot, depicting O₂ concentrations in air-saturated bath solution (black), at the ventricular floor (red), and within the hindbrain (blue) in controls, in EtOH-fixated and KCN-treated preparations; note that EtOH-fixation and bath-application of KCN cause a significant increase of ventricular O₂ concentrations to bath Ringer levels (***p < 0.0001; Mann-Whitney U test). h Scatter plot depicting coinciding ventricular and bath O₂ levels in metabolically inactive (black dots, EtOH-fixated) or oxidative phosphorylation-impaired (pink dots, KCN) preparations. O₂ levels in b–f are color-coded from blue (0 μmol/l) to red (300 μmol/l) to yellow (600 μmol/l); transverse hindbrain schemes indicate motion (c) or position (e, f) of the O₂ electrode. OT, optic tectum; R, rostral; C, caudal; SC, spinal cord; N, number of preparations

Fig. 2.

Influence of bath O₂ concentrations on ventricular O₂ levels. a Recording of O₂ levels in the bath and ventricle following stepwise increase of the bath O₂ concentration from ~ 290 μmol/l (air-saturated) to ~ 550 μmol/l (light gray area), ~ 750 μmol/l (gray area), and ~ 950 μmol/l (dark gray area); the O₂-electrode was initially advanced to the ventricular floor in 0.2 mm steps (left in a) and transiently repositioned to 1.2 mm above the floor (*) prior to each increase of the bath O₂ concentration. b O₂ concentration profiles (mean ± SEM) of ventricular depth tracks (see insets) in air-saturated (~ 290 μmol/l) bath solution (1; N = 31) and after the increase of the bath O₂ concentration to ~ 550 μmol/l (2; N = 6), ~ 750 μmol/l (3; N = 6), and ~ 950 μmol/l (4; N = 6). c Scatter plot, depicting the dependency of the ventricular O₂ concentration (black and red dots; n = 97 from 24 preparations) and adjacent hindbrain (blue dots; n = 69 from 11 preparations) from bath O₂ levels; red dots represent the mean ± SEM of the ventricular O₂ level at distinct bath O₂ concentrations and the red dashed lines linear regressions through the lower (r² = 0.98) and higher (r² = 0.96) range of mean ventricular concentrations, respectively. d, e Scatter plot (d) and boxplot (e) depicting ventricular O₂ consumption as function of the bath O₂ level for concentrations > 700 μmol/l (n = 90 from 13 preparations) with a mean ± SEM of 626 ± 13 μmol/l (e); the slope of the regression line in d (r² = 0.007) is not significantly different from zero (p = 0.42). O₂ levels in a and b are color-coded from blue (0 μmol/l) to red (300 μmol/l) to yellow (600 μmol/l) to green (750 μmol/l) to cyan (900 μmol/l); transverse hindbrain schemes indicate motion (a) or position (b, c) of the O₂ electrode. N, number of preparations; n, number of measurements

Fig. 3.

Correlation between neuronal discharge and ventricular O₂ concentration. a Recording of O₂ levels in the bath (top black trace) and ventricle (green trace) along with the resting rate (red trace) of superior oblique nerve spike activity illustrated at extended time scales (black traces) for selected periods before (before), after spiking has been blocked by bath-applied (0.5%; pink area) MS-222 (no spike activity), and during recovery (recovery); the O₂ consumption (blue trace) was calculated as the difference of concurrent bath and ventricular O₂ concentrations. b Boxplot, depicting O₂ concentrations in the bath (black) and ventricle in controls (red; N = 31) and after bath-application of 0.05% (green; N = 5) and 0.5% MS-222 (blue; N = 6) indicating significantly increased ventricular O₂ levels when the spike activity was blocked (**p < 0.001; ***p < 0.0001; Mann-Whitney U test. c O₂ concentration profile (mean ± SEM) of a midline depth track above the floor of the IV^th ventricle in controls and after bath-application of 0.05% and 0.5% MS-222. d O₂ consumption (upper blue trace), calculated from O₂ concentrations of the bath Ringer and ventricle and corresponding superior oblique nerve discharge rate (upper red trace) during and after bath application of 0.05% MS-222 (pink area); lower traces illustrate the O₂ consumption (blue) and resting rate (red) during the recovery from the drug effect at an extended time and amplitude scale (gray area from above). e Scatter plot, depicting the dependency of calculated O₂ consumption on the resting rate of the superior oblique nerve during recovery from a 0.5% MS-222-induced block of spike activity (N = 6); data from individual preparations are color-coded; each linear regression line has a slope that is significantly different from zero (p < 0.0001); green (r² = 0.52), blue (r² = 0.42), red (r² = 0.40), black (r² = 0.71), pink (r² = 0.55), and orange (r² = 0.69). N, number of preparations

Fig. 4.

Decrease of ventricular O₂ concentrations during spontaneous spike bursts. a Typical multi-unit recording of the superior oblique nerve (black trace) and corresponding firing rate (red trace; bin width 1 s), depicting a spontaneously occurring spike burst (a₁). a₂ shows the transiently elevated discharge rate (red trace) of the multi-unit spike activity in a₁ (gray area) and concurrent alteration in ventricular O₂ concentration (green trace) in an air-saturated bath solution. b Overlay of four consecutive spike burst episodes in an individual preparation (gray traces in the upper plot) and concurrent increases in ventricular O₂ consumption (gray traces in the lower plot) along with the respective averages (red and blue traces)

Fig. 5.

Systematic correlation between neuronal activity and ventricular O₂ levels. a Superior oblique nerve firing rate profile (red trace) of four successive spike bursts (red *) and concurrent alterations of the ventricular O₂ concentration (green trace and *) at a ventricular O₂ level of ~ 100 μmol/l. b Overlay of spike burst episodes (n = 10) from one preparation and corresponding increases in ventricular O₂ consumption (gray traces in the upper and lower plot) along with the respective averages (red and blue traces); vertical dashed line and red arrow indicate the latency of the O₂ transient relative to spike burst onset. c Latency of O₂ transients (n = 69 from 10 preparations) as function of ventricular O₂ concentrations; the slope of the linear regression was not significantly different from zero (r² = 0.004; p = 0.61). d Integrals of spike bursts and concurrent O₂ transients (light red and light green areas in d₁, respectively) at a lower (left in d₁) and a higher ventricular O₂ level (right in d₁; green dashed line); d₂ shows the dependency of spike burst (red circles) and O₂ transient integrals (green circles) on ventricular O₂ levels (n = 30 from 10 preparations); note that the integrals of the spike bursts were independent from (red dashed line; r² = 0.03; p = 0.38), whereas those of the O₂ transients significantly increased (green dashed line; r² = 0.37; p < 0.001) with higher ventricular O₂ levels. e, f Dependency of the ratio of O₂ consumption and spike burst integral (e; n = 30 from 10 preparations) and of the O₂ recovery time (f) from ventricular O₂ levels (n = 13 from 6 preparations); note that the slope of the linear regression of the O₂/firing rate (e) was significantly different from zero (r² = 0.14; p < 0.05), whereas that of the O₂ recovery (f) was not (r² = 0.18; p = 0.15). n, number of measurements