Energy substrates that fuel fast neuronal network oscillations

Abstract

Fast neuronal network oscillations in the gamma-frequency band (30--100 Hz) provide a fundamental mechanism of complex neuronal information processing in the hippocampus and neocortex of mammals. Gamma oscillations have been implicated in higher brain functions such as sensory perception, motor activity, and memory formation. The oscillations emerge from precise synapse interactions between excitatory principal neurons such as pyramidal cells and inhibitory GABAergic interneurons, and they are associated with high energy expenditure. However, both energy substrates and metabolic pathways that are capable to power cortical gamma oscillations have been less defined. Here, we investigated the energy sources fueling persistent gamma oscillations in the CA3 subfield of organotypic hippocampal slice cultures of the rat. This preparation permits superior oxygen supply as well as fast application of glucose, glycolytic metabolites or drugs such as glycogen phosphorylase inhibitor during extracellular recordings of the local field potential. Our findings are: (i) gamma oscillations persist in the presence of glucose (10 mmol/L) for greater than 60 min in slice cultures while (ii) lowering glucose levels (2.5 mmol/L) significantly reduces the amplitude of the oscillation. (iii) Gamma oscillations are absent at low concentration of lactate (2 mmol/L). (iv) Gamma oscillations persist at high concentration (20 mmol/L) of either lactate or pyruvate, albeit showing significant reductions in the amplitude. (v) The breakdown of glycogen significantly delays the decay of gamma oscillations during glucose deprivation. However, when glucose is present, the turnover of glycogen is not essential to sustain gamma oscillations. Our study shows that fast neuronal network oscillations can be fueled by different energy-rich substrates, with glucose being most effective.

Keywords: brain energy metabolism; electrophysiology; glycogen phosphorylase; information processing; lactate; mitochondria; monocarboxylate transporter; synaptic transmission.

Figure 1

Gamma oscillations in slice cultures. (A) Gamma oscillations were induced by bath application of acetylcholine (2 μmol/L) and physostigmine (400 nmol/L) (upper sample trace), and they persisted for more than 60 min (n = 13, N = 6). Gamma oscillations are shown with a higher temporal resolution after 15 min (1), 30 min (2), 45 min (3), and 60 min (4) (lower sample traces). Local field potentials (LFP) were recorded in stratum pyramidale of the CA3 subfield in organotypic hippocampal slice cultures of the rat. (B) Corresponding power spectra of sample traces shown in (A) were calculated from 100 s taken at the end of each data segment. (C) Gamma oscillations were analyzed for various parameters, i.e., peak frequency (Freq), area under curve (AUC), full width at half maximum (FWHM), peak power spectral density (PSD), amplitude (Ampl), and variance of the amplitude (Var). Friedman repeated-measures ANOVA on ranks and Tukey post-hoc test. Statistical significance is marked by asterisks (P < 0.05).

Figure 2

Gamma oscillations and maturation of slice cultures. (A) Slice cultures were stained with toluidine blue after 7, 14, and 21 DIV (left). Gamma oscillations were induced by bath application of acetylcholine (2 μmol/L) and physostigmine (400 nmol/L) and local field potentials (LFP) were recorded in stratum pyramidale of the CA3 subfield (middle). Corresponding power spectra of the sample traces were calculated from data segments of 100 s (right). (B) Gamma oscillations were analyzed for various parameters, i.e., peak frequency (Freq), area under curve (AUC), full width at half maximum (FWHM), peak power spectral density (PSD), amplitude (Ampl) and variance of the amplitude (Var) (7 DIV, n = 12, N = 4; 14 DIV, n = 7, N = 3; 21 DIV, n = 6, N = 3). One-Way ANOVA and Kruskal-Wallis ANOVA on ranks.

Figure 3

Gamma oscillations at low glucose concentration. (A) Gamma oscillations were induced by bath application of acetylcholine (2 μmol/L) and physostigmine (400 nmol/L) in the presence of 10 mmol/L glucose (red). Then, glucose was lowered to 2.5 mmol/L in the recording solution and the properties of gamma oscillations were analyzed after 15 min (black), 30 min (gray), and 45 min (white). Local field potentials (LFP) were recorded in stratum pyramidale of the CA3 subfield (sample traces). (B) Corresponding power spectra of sample traces shown in (A) were calculated from 100 s taken at the end of each data segment. (C) Gamma oscillations were analyzed for various parameters, i.e., peak frequency (Freq), area under curve (AUC), full width at half maximum (FWHM), peak power spectral density (PSD), amplitude (Ampl), and variance of the amplitude (Var) (n = 14, N = 3). Note the decrease in amplitude at low glucose concentration. Repeated-measures ANOVA and Holm-Sidak post-hoc test or Friedman repeated-measures ANOVA on ranks and Tukey post-hoc test. Statistical significance is marked by asterisks (P < 0.05).

Figure 4

Gamma oscillations in the presence of either lactate or pyruvate. (A) Gamma oscillations were induced by bath application of acetylcholine (2 μmol/L) and physostigmine (400 nmol/L). Initially, the recording solution contained 10 mmol/L glucose. Subsequently, recording solutions containing 0 mmol/L glucose (15 min), 2 mmol/L lactate (20 min), and again 10 mmol/L glucose were applied (n = 15, N = 3), while local field potentials (LFP) were recorded in stratum pyramidale of the CA3 subfield (sample traces). (B) Corresponding power spectra of sample traces shown in (A) were calculated from 100 s taken at the end of each data segment. (C) Sample traces of gamma oscillations in the presence of 10 mmol/L glucose vs. 20 mmol/L pyruvate (upper traces) or 20 mmol/L lactate (lower traces), according to the protocol given in (D). (E) Corresponding power spectra of sample traces shown in (C) were calculated from data segments of 100 s. (F) Gamma oscillations were analyzed for various parameters, i.e., peak frequency (Freq), area under curve (AUC), full width at half maximum (FWHM), peak power spectral density (PSD), amplitude (Ampl), and variance of the amplitude (Var) (control 1 and pyruvate, n = 12, N = 4; control 2, and lactate, n = 13, N = 4). Note the significant decrease in amplitude even at the high concentration of lactate or pyruvate. Paired t-test or Wilcoxon signed rank test. Statistical significance is marked by asterisks (P < 0.05).

Figure 5

Gamma oscillations in the presence of lactate and glutamine. (A) Gamma oscillations were induced by bath application of acetylcholine (2 μmol/L) and physostigmine (400 nmol/L) in the presence of 20 mmol/L lactate (black bar); after 30 min, 2 mmol/L glutamine was added (green bar). Local field potentials (LFP) were recorded in stratum pyramidale of the CA3 subfield (sample traces). (B) Corresponding power spectra of sample traces shown in (A) were calculated from 100 s taken at the end of each data segment. (C) Gamma oscillations were analyzed for various parameters, i.e., peak frequency (Freq), area under curve (AUC), full width at half maximum (FWHM), peak power spectral density (PSD), amplitude (Ampl), and variance of the amplitude (Var) (n = 19, N = 4). Note that glutamine has only a minor effect on the frequency of gamma oscillations. Paired t-test. Statistical significance is marked by asterisks (P < 0.05).

Figure 6

Gamma oscillations and glycogen stores during glucose deprivation. (A) Gamma oscillations were induced by bath application of acetylcholine (2 μmol/L) and physostigmine (400 nmol/L) in the presence of 10 mmol/L glucose (white bar). Subsequently, inhibitors of glycogen phosphorylase, DAB (50 μmol/l or 100 μmol/L) or CP-316819 (10 μmol/L or 20 μmol/L) were applied, in the presence (black bar) or absence (light blue bar) of glucose. Note that the standard gas mixture (95% O2 and 5% CO2) was continuously present. Local field potentials (LFP) were recorded in stratum pyramidale of the CA3 subfield subfield (sample trace). (B) The peak power spectral density (μV²/Hz) for each recording trace is shown in black (scaling on left y-axis), the average of all recordings is shown in blue (scaling on right y-axis). Power spectra were calculated every 10 s and plotted over time. (C) The points in time are given for complete suppression of gamma oscillations, i.e., power reaching a threshold defined as the mean of the last 100 s plus 1 standard deviation, according to the protocol given in (A) (control, n = 10, N = 3; DAB, n = 6, N = 3, and n = 5, N = 3; CP-316819, n = 6, N = 3, and n = 5, N = 2). Note that inhibition of glycogen phosphorylase accelerates the decay of gamma oscillations during glucose deprivation. Kruskal Wallis ANOVA on ranks. Statistical significance vs. control is marked by asterisks (P < 0.05).

Figure 7

Gamma oscillations and glycogen turnover. (A) Gamma oscillations were induced by bath application of acetylcholine (2 μmol/L) and physostigmine (400 nmol/L) in the presence of 10 mmol/L glucose. After 15 min, inhibitors of glycogen phosphorylase, DAB (50 μmol/l or 100 μmol/L) or CP-316819 (10 μmol/L or 20 μmol/L) were added to the recording solution. Local field potentials (LFP) were recorded in stratum pyramidale of the CA3 subfield (sample traces). (B) Corresponding power spectra of sample traces shown in (A) were calculated from 100 s taken at the end of each data segment. (C) Gamma oscillations were analyzed for various parameters, i.e., peak frequency (Freq), area under curve (AUC), full width at half maximum (FWHM), peak power spectral density (PSD), amplitude (Ampl), and variance of the amplitude (Var) (control 1 and DAB, n = 13, N = 4; control 2 and CP-316819, n = 10, N = 3). Note that inhibition of glycogen phosphorylase in the presence of 10 mmol/L glucose has only minor effects on gamma oscillations. Paired t-test or Wilcoxon signed rank test. Statistical significance is marked by asterisks (P < 0.05).