Lactate uptake contributes to the NAD(P)H biphasic response and tissue oxygen response during synaptic stimulation in area CA1 of rat hippocampal slices

Abstract

Synaptic train stimulation (10 Hz x 25 s) in hippocampal slices results in a biphasic response of NAD(P)H fluorescence indicating a transient oxidation followed by a prolonged reduction. The response is accompanied by a transient tissue PO(2) decrease indicating enhanced oxygen utilization. The activation of mitochondrial metabolism and/or glycolysis may contribute to the secondary NAD(P)H peak. We investigated whether extracellular lactate uptake via monocarboxylate transporters (MCTs) contributes to the generation of the NAD(P)H response during neuronal activation. We measured the effect of lactate uptake inhibition [using the MCT inhibitor alpha-cyano-4-hydroxycinnamate (4-CIN)] on the NAD(P)H biphasic response, tissue PO(2) response, and field excitatory post-synaptic potential in hippocampal slices during synaptic stimulation in area CA1 (stratum radiatum). The application of 4-CIN (150-250 micromol/L) significantly decreased the reduction phase of the NAD(P)H response. When slices were supplemented with 20 mmol/L lactate in 150-250 micromol/L 4-CIN, the secondary NAD(P)H peak was restored; whereas 20 mmol/L pyruvate supplementation did not produce a recovery. Similarly, the tissue PO(2) response was decreased by MCT inhibition; 20 mmol/L lactate restored this response to control levels at all 4-CIN concentrations. These results indicate that lactate uptake via MCTs contributes significantly to energy metabolism in brain tissue and to the generation of the delayed NAD(P)H peak after synaptic stimulation.

Fig. 1

Series of images taken before, during, and after the stimulus train. (a) Un-subtracted image indicates the region of interest (ROI) within the stratum radiatum (SR) of the CA1 region. The recording electrode is indicated by the white asterisk. Scale bar = 500 μm. Note: The ROI is situated between the stimulating and recording electrodes. Stratum pyramidale (SP) and stratum oriens (SO) are also indicated. (b) Control: NAD(P)H biphasic response consisted of a brief decrease in NAD(P)H fluorescence (oxidation phase) (10 s), followed by a more prolonged NAD(P)H fluorescence increase (reduction phase) (45 s). (c) α-Cyano-4-hydroxycinnamate (4-CIN): Effect of monocarboxylate transporter inhibition by 4-CIN on the NAD(P)H biphasic response. Slices were incubated with 4-CIN 15 min prior to the stimulus train. In the presence of monocarboxylate transporter blocker 4-CIN (150 μmol/L), the early oxidation (10 s) was not affected, while the secondary NAD(P)H fluorescence peak was significantly decreased (45–80 s).

Fig. 2

Representative traces showing changes in NAD(P)H fluorescence, tissue PO₂, and excitatory post-synaptic potential (EPSP) amplitude after brief synaptic stimulation. Slices were incubated with α-cyano-4-hydroxycinnamate (4-CIN; 15 min) with or without lactate (10 min) prior to the stimulus train. (a) In the presence of 4-CIN (250 μmol/L) the late NAD(P)H peak was reduced, but was restored by lactate supplementation. (b) The transient decrease in tissue PO₂ occurring during the stimulus train was reduced by 30% with 4-CIN and restored to control values by lactate supplementation. (c) 4-CIN and lactate did not significantly affect field EPSP amplitude. [Correction added after online publication (18/10/07): at the top of the figure 25 Hz was changed to 10 Hz].

Fig. 3

Monocarboxylate transport inhibition by α-cyano-4-hydroxycinnamate (4-CIN) results in the decrease of the NAD(P)H biphasic response. Various concentrations of 4-CIN were applied to the hippocampal slice for 15 min before the stimulus train. (a) Representative traces of the NAD(P)H biphasic response before, during, and after the application of monocarboxylate transporter inhibitor 4-CIN (250 μmol/L). (b) The effect of 4-CIN on the NAD(P)H biphasic response is concentration dependent. The application of 150–250 μmol/L 4-CIN significantly decreased the reduction phase of the NAD(P)H response while the oxidation phase was not affected. At higher concentrations (i.e. 500 μmol/L) both reduction and oxidation were significantly decreased. Data are the mean ± SEM of 5–18 slices/condition. ***p < 0.001 and **p < 0.01 versus control (ANOVA and Tukey's multicomparison test).

Fig. 4

Effect of monocarboxylate transporter inhibitor α-cyano-4-hy-droxycinnamate (4-CIN) on the tissue PO₂ response. Various concentrations of 4-CIN were applied to the hippocampal slice for 15 min before the stimulus train. Twenty minutes after 4-CIN was removed from the perfusion buffer the stimulus train was repeated to demonstrate that the effect of 4-CIN on the tissue PO₂ response was reversible. (a) Representative traces of the tissue PO₂ response before, during, and after the application of monocarboxylate transporter inhibitor 4-CIN (250 μmol/L). In the presence of 4-CIN, the PO₂ tissue response during a stimulus train is reduced. The arrows (↓) indicate when the stimulus train started. (b) The effect of 4-CIN, on the tissue PO₂ response, was concentration dependent. Data are the mean ± SEM of 4–12 slices/condition. ***p < 0.001 and **p < 0.01 versus control (ANOVA and Tukey's multicomparison test).

Fig. 5

Hippocampal slices were supplemented with lactate (10–20 mmol/L) 10 min before the stimulus train (10 mmol/L glucose). Lactate supplementation alone did not change the amplitude of the NAD(P)H biphasic response (a) or the amplitude of tissue PO₂ response (b). Data are the mean ± SEM of four to six slices, NS p > 0.05 (ANOVA and Tukey's multicomparison test).

Fig. 6

Effect of lactate supplementation on the NAD(P)H biphasic response and the tissue PO₂ response in the presence of α-cyano-4-hydroxycinnamate (4-CIN). After synaptic stimulation hippocampal slices were supplemented with lactate (20 mmol/L) 10 min before the stimulus train in the presence of monocarboxylate transporter inhibitor 4-CIN. (a) Lactate restored the reduction phase of the NAD(P)H response in the presence of 4-CIN at lower concentrations. The early NAD(P)H oxidation phase was decreased only in the presence of 500 μmol/L 4-CIN and was restored to control levels by lactate supplementation. Data are the mean ± SEM of 5-35 slices. ***p < 0.001 versus control, ^†p < 0.05 versus 4-CIN (anova and Tukey's multicomparison test). (b) Lactate supplementation restored tissue PO₂ response to control levels at all 4-CIN concentrations. Data are the mean ± SEM of 4-13 slices. **p < 0.01, ***p < 0.001 versus control, ^†p < 0.05 versus 4-CIN (anova and Tukey's multicomparison test).

Fig. 7

Effect of hypoglycemia and lactate/pyruvate substitution on the NAD(P)H biphasic response. Slices were incubated with 2.5 mmol/L glucose for 30 min before the stimulus train and were returned to 10 mmol/L glucose after 60 min of exposure to hypoglycemia. In some cases, slices were supplemented with pyruvate or lactate (20 mmol/L) during hypoglycemia. Data are the mean ± SEM of 4–13 slices. *p < 0.05 versus control (ANOVA and Tukey's multicomparison test).