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Keywords:

  • brain slices;
  • cortex;
  • energy substrates;
  • GABA;
  • hippocampus;
  • network oscillations

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

J. Neurochem. (2009) 112, 900–912.

Abstract

While the ultimate dependence of brain function on its energy supply is evident, how basic neuronal parameters and network activity respond to energy metabolism deviations is unresolved. The resting membrane potential (Em) and reversal potential of GABA-induced anionic currents (EGABA) are among the most fundamental parameters controlling neuronal excitability. However, alterations of Em and EGABA under conditions of metabolic stress are not sufficiently documented, although it is well known that metabolic crisis may lead to neuronal hyper-excitability and aberrant neuronal network activities. In this work, we show that in slices, availability of energy substrates determines whether GABA signaling displays an inhibitory or excitatory mode, both in neonatal neocortex and hippocampus. We demonstrate that in the neonatal brain, Em and EGABA strongly depend on composition of the energy substrate pool. Complementing glucose with ketone bodies, pyruvate or lactate resulted in a significant hyperpolarization of both Em and EGABA, and induced a radical shift in the mode of GABAergic synaptic transmission towards network inhibition. Generation of giant depolarizing potentials, currently regarded as the hallmark of spontaneous neonatal network activity in vitro, was strongly inhibited both in neocortex and hippocampus in the energy substrate enriched solution. Based on these results we suggest the composition of the artificial cerebrospinal fluid, which bears a closer resemblance to the in vivo energy substrate pool. Our results suggest that energy deficits induce unfavorable changes in Em and EGABA, leading to neuronal hyperactivity that may initiate a cascade of pathological events.

Abbreviations used:
ACSF

artificial CSF

APV

2-amino-5-phosphovalerate

BCECF

bis(carboxyethyl)-5(and-6)-carboxyfluorescein

BHB

beta-hydroxybutyrate

DFGABA

driving force for GABA-induced currents

eACSF

enriched energy substrates ACSF

EGABA

reversal potential of GABA-induced anionic currents

Em

resting membrane potential

ES

energy substrates

GDPs

giant depolarizing potentials

KB

ketone body

NBQX

2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline

sIPSC

spontaneous inhibitory postsynaptic current

Energy homeostasis, like any physiological control system, is a multilayer arrangement permeating all levels of organization from molecular to behavioral. A key component of this system is energy substrate availability; the sufficient supply of one or more energy substrates (ES) and an adequate means to utilize those substrates (Prins 2008).

In the brain, the ES pool is predominantly composed of glucose, ketone bodies (KBs), pyruvate, and lactate (Nehlig 1997; Lust et al. 2003; Erecinska et al. 2004; Prins 2008). The exact proportion of each substrate in the energy pool varies depending on the species, physiological demands, and energy substrate availability and this variability is especially prominent in the immature developing brain (Booth et al. 1980; Bougneres et al. 1986; Herrera 2002; Erecinska et al. 2004; Nehlig 2004; Ward Platt and Deshpande 2005).

During gestation energy supply to the developing foetus occurs by the transplacental passage of glucose, amino acids, and fatty acids from the mother. This placental support mechanism provides all the necessary energy and cofactors for normal fetal development. At birth this transplacental supply of nutrients ends and crucial changes in the energy supply occur. Following a brief pre-suckling period (postnatal starvation) there is an adaptation to a fat-rich diet (Girard et al. 1992; Medina and Tabernero 2005; Ward Platt and Deshpande 2005). Immediately after birth but before suckling, KBs are not available and lactate is the main energy substrate to the newborn (Girard et al. 1992; Medina et al. 1996; Medina and Tabernero 2005; Ward Platt and Deshpande 2005). The rate of lactate utilization by neurons in the early neonatal rat brain is significantly higher than that of glucose or beta-hydroxybutyrate [BHB, the predominant ketone body in the blood (Bough and Rho 2007)] (Arizmendi and Medina 1983; Fernandez and Medina 1986; Vicario et al. 1991) and recent results showed the importance of lactate as a cerebral oxidative energy substrate (Schurr and Payne 2007; Bak et al. 2009; Castro et al. 2009).

In the postnatal developing rat brain, blood glucose levels are close to those in adults (Pereira de Vasconcelos and Nehlig 1987; Nehlig and Pereira de Vasconcelos 1993). However, glucose utilization is limited and is only about 20% of adult levels (Nehlig et al. 1988; Dombrowski et al. 1989; Vannucci and Vannucci 2000). Indeed, until maturation of the enzymatic systems required for glucose oxidation is complete (Land et al. 1977; Leong and Clark 1984b; Dombrowski et al. 1989) even high concentrations of glucose fail to provide adequate energy for neonatal neurons. During this period other ES in addition to glucose (additional ES), including KB and lactate, make up the energy substrate balance. Indeed, the immature rat brain utilizes KBs so effectively that they contribute more than 30% to the ES pool (Nehlig 2004).

Surprisingly, while the developing brain uses different ES at different developmental stages, artificial CSF (ACSF) used for in vitro experiments is standard for all ages and contains glucose as the sole energy substrate. The absence of additional ES to fully meet the demands of the developing brain may result in an acute energy deficit in brain slices and may affect energy-sensitive processes within neurons and compromise function of neurons and neuronal networks. In this study we focus on the neuronal responses to variations in additional ES delivery to the neonatal brain, and the effects of energy substrate availability on: (i) basic parameters controlling neuronal excitability [the resting membrane potential (Em) and the reversal potential of GABA-induced currents (EGABA)]; (ii) synaptic GABA signaling and (iii) spontaneous neonatal neuronal network activity, giant depolarizing potentials (GDPs). Numerous studies have reported strongly depolarizing, excitatory GABA signaling and the presence of GDPs as characteristic features of developing neuronal networks in vitro (Ben-Ari et al. 2007; Galanopoulou 2007; Kahle and Staley 2008). We found, however, that GDP generation, and a depolarizing shift in both Em and GABA signaling occur when immature neocortical and hippocampal neurons experience periods of energy substrate availability deficiency.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Slice recordings

Brain slices were prepared from postnatal day P3–P8 Swiss mice and Wistar rats of both sexes. All animal protocols conformed to the French Public Health Service policy and the INSERM guidelines on the use of laboratory animals. Animals were rapidly decapitated and brains removed. Sagittal slices (300 μm) were cut using a tissue slicer (Microm International, Walldorf, Germany) in ice-cold oxygenated modified artificial cerebrospinal fluid, with 0.5 mM CaCl2 and 7 mM MgSO4, in which Na+ was replaced by an equimolar concentration of choline. Slices were then transferred to oxygenated standard ACSF containing (in mM): 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 25 NaHCO3, 1.3 MgCl2, 2.0 CaCl2, and 10 d-glucose, pH 7.4, at room temperature (20–22°C) for at least 1 h before use. During recordings, slices were placed in a conventional fully submerged chamber superfused with ACSF (32–34°C). Pyramidal cells were identified by the characteristic morphology of their soma, and the presence of a prominent apical dendrite, using IR-differential interference contrast video microscopy. Pyramidal cells in both deep and superficial neocortical layers and CA1 and CA3 hippocampal regions were recorded. Patch-clamp recordings were performed using dual EPC-10 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany). Pipettes (resistance of 3.5–8 MΩ) were pulled from borosilicate glass capillaries.

The ES combinations used in experiments

The following ES solutions were used: Glucose-BHB; Glucose-pyruvate; Glucose-lactate; Glucose-BHB-pyruvate [enriched energy substrates ACSF (eACSF)] and Glucose-BHB-lactate (lactate-based eACSF). We suggest that the eACSF solution (termed eACSF – as it supplies sufficient energy substrates to the brain slices) may be a suitable alternative to standard ACSF for the reasons discussed below. In all combinations, ES were used in the following concentrations (in mM): 5 glucose, 5 Na-pyruvate, 5 Na-lactate, 2 BHB. The eACSF solution contained (in mM): 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 25 NaHCO3, 1.3 MgCl2, 2.0 CaCl2, 5 choline chloride, 5 glucose, 2 BHB and 5 Na-pyruvate, pH 7.4 (305 mosm).

About 5 mM Na-lactate was used instead of Na-pyruvate when noted. Choline chloride was used in this study solely to allow comparison with our standard ACSF solution (osmolarity, Na+ and Cl concentrations as in standard ACSF). 5 mM choline chloride can however be replaced by NaCl.

Bicarbonate free solution

The CO2/HCO3-free solutions (termed HCO3-free solution further in the text) contained (in mM): 126 NaCl, 3.5 KCl, 2 CaCl2, 1.3 MgCl2, 25 Na_HEPES (pH 7.4), plus ES relevant for the particular experiment, and the solutions were bubbled with O2.

Patch clamp recordings in cell-attached configuration

The currents through NMDA channels reverse close to 0 mV, therefore in cell-attached recordings, measurement of the reversal potential for NMDA receptor channels gives the value for Em (for more details see Tyzio et al. 2003, 2008). Meanwhile measurement of the reversal potential of the GABA receptor channels provides the driving force (DFGABA) for the GABA induced currents (for more details see Rheims et al. 2008).

Single channel recordings were performed using a pipette solution containing (in mM): (i) for recordings of single GABA channels: NaCl 140, KCl 2.5, CaCl2 2, MgCl2 1, HEPES 10, GABA 0.01, pH adjusted to 7.3 with NaOH; (ii) for recordings of single NMDA channels: 140 NaCl, 2.5 KCl, 2 CaCl2, 10 HEPES, 0.01 NMDA, 0.01 glycine, pH adjusted to 7.3 with NaOH. Analysis of currents through single channels was performed using IGOR-Pro software (WaveMetrics, Inc., Lake Oswego, OR, USA). All DFGABA values were corrected for −2.1 mV (Tyzio et al. 2008).

Gramicidin patch recordings

Gramicidin-d (50 μg/mL) perforated patch recordings were performed as described previously (Tyzio et al. 2007). Glutamatergic transmission was blocked by bath application of 2-amino-5-phosphovalerate (APV; 40 μM) and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX; 10 μM). For evoked responses nerve fibers were stimulated (pulse duration 0.2 ms) each 20 s via a bipolar metal electrode positioned at a distance of about 50–100 μm from the recording site.

Fluorescence pH measurements

Prior to measurements brain slices were incubated for 30–40 min in 10 μM 2′,7′-bis(carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF), acetoxymethyl ester form (Molecular Probes, Eugene, OR, USA) dissolved in standard ACSF solution at 32–34°C. Fluorescence images were acquired using a customized digital imaging microscope. Excitation of cells at 440 and 490 nm wavelengths was achieved using a 1 nm bandwidth polychromatic light selector equipped with a 100 W xenon lamp (Polychrome II; TILL Photonics, Gräfelfing, Germany). Light intensity was attenuated using neutral density filters. A dichroic mirror (495 nm; Omega Optical, Brattleboro, VT, USA) was used to deflect light onto the samples. Fluorescence was visualized using an upright microscope (Axioskop; Zeiss, Göttingen, Germany) equipped with an infinity-corrected 60× water-immersion objective (n.a. = 0.9; LumPlanFL; Olympus, Center Valley, PA, USA). Fluorescent-emitted light passed to a 16 bit electron multiplying charge-coupled device digital camera system (Andor iXon EM+; Andor Technology PLC, Belfast, Northern Ireland). Fluorescence signals from the BCECF loaded neocortical pyramidal cells were acquired using Andor iQ software (Andor Technology PLC). The average fluorescence intensity of each region of interest was measured. Mean background fluorescence (measured from a non-fluorescent area) was subtracted and the ratio intensities (F490/F440) were determined. The duration of excitation was 10–50 ms (sampling rate 0.1 Hz).

For calibration of BCECF the K+/H+ ionophore nigericin (10 μM) was used to equilibrate extra- and intracellular pH. The external solution contained (in mM): 140 KCl, 2 CaCl2, 2 MgCl2, 10 d-glucose, buffered by 20 HEPES (adjusted with NaOH to different pH values). Solutions with 6.05, 7.0, 7.2, 7.4 and 8.0 pH values were used.

Pharmacology

Drugs used were purchased from Tocris (Cookson, Ballwin, MO, USA) (NBQX, d-APV) and Sigma (St Louis, MO, USA) (racemic mixture of BHB; dl-3-hydroxybutyric acid sodium salt, pyruvate sodium salt, lactate sodium salt, Gramicidin-d). Within the racemic mixture, d-BHB is the primary mediator of the physiological effects of dl-BHB, and is the only form that can function as a substrate for mitochondrial BHB dehydrogenase (Klee and Sokoloff 1967; Passingham and Barton 1975; Robinson and Williamson 1980; Eaton et al. 2003). Consequently, only 50% of exogenous dl-BHB is expected to be utilized (Tsai et al. 2006).

Statistical analysis

Group measures were expressed as means ± SEM; error bars also indicate SEM. Statistical significance was assessed using the Wilcoxon’s signed rank test, the Mann–Whitney U test and Kruskal-Wallis non-parametric anova test. The level of significance was set at p < 0.05. In the figures, one star corresponds to p < 0.05, two stars corresponds to p < 0.01, three stars corresponds to p < 0.001).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Effects of energy substrates on Em and EGABA in cortical pyramidal cells

Previously we have shown in neocortical neurons in slices that BHB can induce a shift in neuronal Em and EGABA (Rheims et al. 2009). To determine whether these effects are particular to BHB or whether other ES also induce similar changes we examined the effects of different ES added to the external solution on Em and EGABA. Initially both parameters were measured in the presence of standard glucose based ACSF. Subsequently, glucose was complemented with either; BHB, pyruvate or lactate, and Em and EGABA were re-measured. Em and EGABA were assessed using cell-attached recordings of single NMDA and GABA receptor channels, respectively (Rheims et al. 2008, 2009). In all experiments, a ‘paired test’ protocol was used (Rheims et al. 2009) and single channels were recorded on the same target cell in control and following at least 40 min of the energy substrate enriched solution application.

Figure 1(a) and (d) shows the paired test results for different ES combinations*. In neocortical pyramidal cells (P3–P8), application of all ES combinations tested induced a significant hyperpolarization of both Em and EGABA. To compare the effects of the different ES combinations the average values of Em and EGABA in each experimental set were normalized to the values in standard ACSF (Fig. 1b and e). Hyperpolarization of Em and EGABA was not significantly different with different ES combinations (p > 0.4 and p > 0.06, respectively) and in Fig. 1(c) and (f) data are pooled to show the effect of all additional ES on Em and EGABA (Em: −75.1 ± 0.9 mV in ACSF vs. −83 ± 0.7 mV with additional ES, n = 49, p < 0.001; EGABA: −53.2 ± 1.7 mV in ACSF vs. −79.4 ± 1.8 mV with additional ES, n = 49, p < 0.001).

image

Figure 1.  Supplementation glucose with additional energy substrates induces hyperpolarization of Em and EGABA. Em and EGABA were evaluated by measuring I–V relations of single NMDA- and GABA-activated channels in the cell-attached configuration in neocortical (panel a–f) and in CA1 and CA3 hippocampal pyramidal neurons (panels g and h) from brain slices (P3–P8 rats). A ‘paired test’ protocol (Rheims et al. 2009) was used in all experiments (a, d, g, h): the left circles in each group depict values measured in ACSF while the right circles depict values measured on the same cells following application of energy substrate enriched solutions. For simplicity, additional ES in figures denote the following compositions (see Materials and Methods): BHB (glucose-BHB solution); pyruvate (glucose-pyruvate solution); lactate (glucose-lactate solution); eACSF (glucose-BHB-pyruvate solution). (b, e) Hyperpolarization of Em and EGABA induced by energy substrate enriched solutions normalized to the values in ACSF. *,**,*** denote the statistical significance with respect to ACSF. (c, f) Pooled Em and EGABA data. (g, h) Em and EGABA measured in CA1 and CA3 hippocampal neurons using a ‘paired test’ protocol. (i) Deviations of the reversal potential of GABA-mediated currents in respect to the resting membrane potential, DFGABA, in different experimental sets (depicted by different colors). Grey-filled circles correspond to values in ACSF. Note that in all cases, GABA signaling changed its mode from a strongly depolarizing in ACSF to a shunting/inhibitory after addition of ES.

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The effects of the energy substrate enriched solutions were not region-specific as they also induced hyperpolarization of Em and EGABA in pyramidal cells of CA1 (Fig. 1g; Em: −74.4 ± 1 mV in ACSF vs. −78.1 ± 3 mV with BHB, n = 6, p < 0.05; EGABA: −65.9 ± 4.8 mV in ACSF vs. −78.8 ± 6.5 mV with BHB, n = 6, p < 0.05) and CA3 (Fig. 1h; Em: −71.7 ± 1.5 mV in ACSF vs. −87 ± 2.1 mV with eACSF, n = 6, p < 0.02; EGABA: −53.6 ± 2.1 mV in ACSF vs. −89.5 ± 4.7 mV with eACSF, n = 6, p < 0.04) hippocampal regions.

Figure 1(i) demonstrates DFGABA values for different experimental sets shown above. In all cases, GABA signaling changed its mode from a significantly depolarizing in ACSF to a shunting/inhibitory after addition of ES [21.5 mV in ACSF vs. 4 mV in BHB; 21.3 mV in ACSF vs. 2.8 mV in pyruvate; 23.6 mV in ACSF vs. 0.1 mV in lactate; 16.7 mV in ACSF vs. −5.7 mV in eACSF; 8.6 mV in ACSF vs. −0.7 mV in BHB (CA1); 18.1 mV in ACSF vs. −1.3 mV in eACSF (CA3)].

Therefore, supplementing glucose with different ES induced similar effects on Em and EGABA of pyramidal neurons: prominent hyperpolarization.

These results also suggest that the effects of BHB on Em and EGABA described in our previous study (Rheims et al. 2009) may be because of the role of ketone bodies as energy substrates. In that paper we showed that BHB mediated regulation of [Cl]i was directly correlated to the activity of HCO3/Cl transporter(s) [some of which have an absolute requirement for the presence of HCO3 in the extracellular media (Romero et al. 2004; Pushkin and Kurtz 2006)]. This mechanism is confirmed in the present study as in neocortical neurons, neither BHB nor pyruvate affected EGABA when HCO3-free solution was used (Fig. 2a and b; BHB: −80.1 ± 2.7 mV in the glucose-BHB solution, n = 28; −56.8 ± 3.1 mV in the HCO3-free solution, n = 9: p > 0.4 compared with ACSF. Pyruvate: −80 ± 2.8 mV in the glucose-pyruvate solution, n = 18; −61.1 ± 2.8 mV in the HCO3-free solution, n = 9: p > 0.05 compared with ACSF). This suggests that at this developmental stage the balance between activity of NKCC1 and HCO3/Cl transporters mostly defines [Cl]i (see Discussion and Fig. 5). Importantly, however, Em values in HCO3-free solution did not differ significantly from those in glucose-BHB and glucose-pyruvate solutions (Fig. 2a and b; BHB: −84.5 ± 0.9 mV in the glucose-BHB solution, n = 28; −83.8 ± 1.4 mV in the HCO3-free solution, n = 9, p > 0.6. Pyruvate: −81.2 ± 1.1 mV in the glucose-pyruvate solution, n = 18; −82.7 ± 2 mV in the HCO3-free solution, n = 9, p > 0.1) indicating that the energy substrate enriched solutions still have a hyperpolarizing action on Em in the absence of bicarbonate.

image

Figure 2.  On the mechanism of energy substrates action. (a, b) Energy substrates do not affect EGABA in the absence of HCO3 but still hyperpolarize Em. Mean values of EGABA and Em determined in glucose-BHB (BHB) (a) or glucose-pyruvate (Pyruvate) solution (b) in the presence (left columns) or absence (right columns) of HCO3. Dashed lines show the average values in ACSF. Note that in the absence of bicarbonate neither BHB nor pyruvate causes changes in EGABA. However, the absence of bicarbonate does not affect the hyperpolarizing action of BHB and pyruvate on Em. (c) In paired test, Em does not differ significantly in eACSF and HCO3-free solution containing similar ES (eHCO3-free). (d, e) Effect of energy substrates on intracellular pH. (d) Calibration curve of the pH sensitive dye BCECF. Fluorescence ratio (F490/F440) for pH values 6.05; 7.0; 7.2; 7.4 and 8.0 were measured. (e) Change in the intracellular pH in a neocortical pyramidal cell during exchange of ACSF for eACSF and during NH4+ (10 mM) application. Note that following substitution of ACSF for eACSF the shift in pHi do not exceed 0.1 pH units.

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image

Figure 5.  Hypothetical scheme summarizing the effects of energy substrates on the neuronal resting potential and intracellular chloride concentration in neonatal neurons. Glucose, lactate, pyruvate and ketone bodies (KBs) are converted into acetyl-coenzyme A, the main input for the citric acid (Krebs) cycle (CAC) leading to energy production. Glucose alone, even at high concentration, cannot maintain mitochondrial respiration at the level necessary for optimal neuronal functioning. Complementing glucose with pyruvate, lactate or ketone bodies allows energy demands to be met. This results in a stimulation of pumps, transporters or channels causing, in particular, a hyperpolarizing shift in resting membrane potential (RMP) and a decrease in intracellular Cl concentration. The contribution of transporters, NKCC1, KCC2 and Cl/HCO3 transporter(s), to the regulation of [Cl]i changes during development. In the early stages of development, the regulation of [Cl]i may be primarily mediated via the interaction between NKCC1 and the Cl/HCO3 transporters. The abbreviations used in this figure were: Acetyl-Co A, acetyl-coenzyme A; CAC, citric acid cycle; RMP, resting membrane potential; NKCC1, Na-2Cl-K cotransporter; KCC2, K-Cl cotransporter.

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To further verify that Em is not affected by the change in [Cl]i we performed a ‘paired test’ on neurons superfused either with eACSF (low [Cl]i) or with HCO3-free solution supplemented with the same energy substrates as in eACSF (high [Cl]i) (Fig. 2c). Values of Em were not significantly different in these solutions (−72.6 ± 0.6 mV in eACSF and −74.6 ± 0.6 mV in HCO3-free solution; n = 17; p > 0.1).

It was suggested (Glykys et al. 2009) that the effects of BHB on GABA signaling (Rheims et al. 2009) may be because of acidification of intracellular pH. Therefore, we tested whether the addition of energy substrates to the extracellular solution significantly alters intracellular pH. Importantly, extracellular pH (7.4) was not affected by the addition of ES. Fluorescence measurements made using the pH-sensitive dye BCECF (Fig. 2c) showed that a 40-min exchange of standard ACSF for eACSF resulted in a decrease in intracellular pH by only 0.05 pH units (Fig. 2d and e; pH −7.3 ± 0.05 in ACSF, pH −7.25 ± 0.05 in eACSF; in each slice (n = 6) values were obtained by averaging signals from 5 to 10 neurons). Meanwhile, in agreement with earlier observations in hippocampal neurons (Schwiening and Boron 1994; Bevensee et al. 1996), 10 mM NH4+ application caused a strong biphasic pH change (Fig. 2d). These observations suggest that the effects of energy substrates on Em and EGABA were not largely dependent on changes in pHi.

Effects of additional ES on GABAergic synaptic activity

While measurements of EGABA using single channel recordings correlate with somatic [Cl]i, values of [Cl]i at dendritic synaptic sites may be different (Gulledge and Stuart 2003). We therefore recorded spontaneous GABAergic synaptic currents (sIPSC; glutamatergic synaptic transmission was blocked by NBQX and APV) in P4–P5 neocortical pyramidal neurons (six experiments) using the gramicidin perforated patch clamp technique to preserve endogenous [Cl]i. Figure 3(a) shows that sIPSCs strongly decreased following the exchange of standard ACSF for eACSF (holding potential, Vh = −100 mV) suggesting a significant shift to more negative values of EGABA at synaptic sites during eACSF application (see Fig. 3b and c). The effect of eACSF was completely reversed after washout with standard ACSF.

image

Figure 3.  eACSF strongly affects GABAergic synaptic transmission. GABAergic synaptic currents recorded from cortical pyramidal neuron using gramicidin perforated patch. Brain slice from P4 rat. Glutamatergic transmission was blocked by NBQX (10 μM) + APV (50 μM). Traces were filtered (0.5 Hz high-pass and 500 Hz low-pass). (a) Spontaneous GABAergic synaptic currents (sIPSC) recorded at a holding potential of −100 mV. (i), (ii), (iii) show single IPSCs at the time-points indicated in the panel above. (b) Recording at −60 mV holding potential in ACSF (top trace) and in eACSF (bottom trace): note that sIPSC were inwardly directed in ACSF and outwardly directed following exchange of ACSF for eACSF. (i), (ii) show examples of postsynaptic currents at extended timescale corresponding to the time-points indicated in the panels above. (c) Examples of evoked GABAergic synaptic currents (other perforated patch). Note that the evoked current was inwardly directed in ACSF, changed directions following exchange of ACSF for eACSF and restored the inward direction following washout of eACSF for ACSF.

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Interestingly, following the application of eACSF there was an outward shift in the baseline current, which presumably correlates with activation of a system regulating Em (Fig. S1). The baseline current returned to its initial level following washout of eACSF with ACSF.

To confirm that eACSF application may cause a significant shift to negative values of EGABA at synaptic sites we depolarized membrane potential by applying Vh = −60 mV and recorded sIPSCs (Fig. 3b). In this case, the membrane potential was presumably slightly more negative than EGABA in ACSF but more positive than EGABA in eACSF. In standard ACSF, sIPSCs had an inward direction, while the current direction was outward in the same cells after exchange of standard ACSF for eACSF (n = 3). A similar effect was observed with evoked GABAergic synaptic responses (Fig. 3c). At the same holding potential (Vh = −60 mV), synaptic currents induced by stimulation of afferent fibers were inward in ACSF, changed their direction to outward in eACSF and returned to inward again following washout of eACSF with ACSF (n = 3).

These results demonstrate that energy substrate enriched solutions strongly affect not only somatic GABA signaling, but also synaptic GABAergic transmission. However, we cannot rule out a possibility that in some dendritic (Gulledge and Stuart 2003) or axonal (Price and Trussell 2006; Trigo et al. 2007; Khirug et al. 2008) compartments [Cl]i regulation may differ from that observed in our experiments.

Effects of energy substrate enriched solutions on spontaneous network activity, GDPs

In standard ACSF, GDPs were reliably recorded both in the neocortex and in the hippocampus of P4–P6 rats and mice (Fig. 4a–d). However, when ACSF was replaced by eACSF there was a complete and reversible elimination of GDP activity in all experiments (n = 5 for neocortex and n = 7 for CA3). When a lactate-based eACSF (eACSF_lactate; pyruvate replaced by 5 mM lactate) was applied during a similar experimental protocol, GDPs were strongly reduced but not completely eliminated in either the neocortex (n = 3) or the hippocampus (n = 3) (Fig. 4c and d). We suggest that after slicing, long-lasting storage of slices in standard ACSF may lead to irreversible changes in some biochemical processes thus partially preventing the conversion of lactate to pyruvate. Indeed, when we exchanged the slicing procedure to one in which slices were transferred to the lactate-based eACSF directly following slicing, GDPs were not observed until the solution was exchanged for ACSF (Fig. 4e and f; n = 2 for neocortex and n = 5 for CA3).

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Figure 4.  GDPs are not generated by neocortical and hippocampal networks in eACSF. Field recordings of spontaneous neuronal network activity in brain slices. Traces were filtered at 1 Hz high-pass and 20 Hz low-pass. (a, b) Effect of exchange of ACSF for eACSF on GDPs generation in neocortex (a) and hippocampus (b). Arrows with horizontal bars indicate regions used for illustration of expanded traces. Note that in standard ACSF, GDPs were readily generated both in neocortical deep layers and CA3. However, they were completely eliminated following exchange of ACSF for eACSF. GDPs re-appeared following washout of eACSF for ACSF. (c, d) In similar experimental protocol, the lactate-based eACSF with pyruvate replaced by lactate did not completely prevent GDP generation. (e, f) However, if slices were placed in the lactate-based eACSF (eACSF_lactate: pyruvate replaced by 5 mM lactate) directly after the cutting procedure, GDPs were not observed.

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Additional to glucose ES differed in their efficacy of inhibiting GDPs. For instance, supplementing glucose with BHB did not considerably inhibit the GDP generation while supplementing glucose with pyruvate prevented the GDP generation completely (data not shown). However, in this study we did not examine in detail this aspect of ES action on spontaneous activity.

If we enhanced neuronal excitability by increasing [K+]o to 5 mM, the oscillatory, GDP-like, network activity re-appeared (Fig. S2). However, whether these oscillations have a similar mechanism of generation to GDPs is as yet unknown.

Altogether these results show that complementing glucose in standard ACSF with ES may strongly suppress the generation of GDPs in slices of neocortex and hippocampus.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In this study, we show that during the postnatal development of the cortex, two critical parameters controlling neuronal excitability, Em and EGABA, can be adversely affected by an inadequate composition of the energy substrate pool. Complementing glucose in standard ACSF with energy substrates has a profound effect on both Em and EGABA: both Em and EGABA become significantly more negative, and GABA action is shunting, or even inhibitory. Our observations also indicate that deficiency in ES availability results in hyperexcitability of cortical neuronal networks.

We showed previously (Rheims et al. 2009) that during postnatal neocortical development Em and EGABA are sensitive to the availability of KBs, which effectively reduce the depolarizing effect of GABA in a dose-dependent manner. Here, we show that this effect is not specific for KBs and is also produced by other ES. Therefore, the action of KBs on neuronal excitability (at least on Em and EGABA) may be explained by their function as energy substrates. The KB mediated effect on [Cl]i correlated with the activity of HCO3/Cl transporter(s) (Rheims et al. 2009). Similar to BHB, pyruvate failed to modulate EGABA in the HCO3-free solution indicating that there is a common mechanism for the regulation of [Cl]i by additional ES. Meanwhile, both BHB and pyruvate induced a hyperpolarizing shift in Em even in the HCO3-free solution. The primary reason for this test was a possible existence of a tonic Cl current (Semyanov et al. 2004) and therefore, a change in [Cl]i would induce a shift in Em. This did not occur in our experimental conditions presumably because of a negligible ambient GABA concentration in slices. It is still possible, however, that in vivo a tonic GABA current may contribute to the control of Em. This suggests that there may be an energy-dependent regulation of Em, which should be investigated in a future study. A possible means by which the different ES can induce similar changes in Em and EGABA is shown in Fig. 5. The relative contribution of NKCC1, KCC2 and the Cl/HCO3 transporters to control of [Cl]i will vary during development. In the immediate postnatal period described in this study, the balance between the actions of NKCC1 and the Cl/HCO3 transporters are likely to determine [Cl]i.

An alternative mechanism that has been suggested is that the effects of additional ES are mediated by intracellular acidification (Glykys et al. 2009). Indeed, weak acids including lactic acid and butyric acid, have been used to induce intracellular acidification (see for review Roos and Boron 1981) but normally at concentrations much higher than those used in our study. For instance, 20 mM lactate (pHo = 7.0) was used to decrease pHi (Kaila et al. 1993) inducing a hyperpolarising shift in the reversal potential of inhibitory postsynaptic potentials (IPSPs) in mature neocortical neurons of about 5.5 mV. In our experiments, the addition of BHB and pyruvate caused only slight acidification of pHi (see Fig. 2e) compared with standard ACSF. Therefore, it is unlikely that intracellular acidification plays a major role in the effects of additional ES on Em and EGABA, although we cannot rule out the possibility of a contribution of alterations in pHi to the changes in [Cl]i. Importantly, it should be noted that whether or not changes in pHi also play a role in the ES meditated modulation of Em and EGABA the presence of a combination of ES in the ACSF is more reflective of the physiological situation.

Previous in vitro studies have reported that GABA is depolarising and excitatory during the first two postnatal weeks of rodent development (for reviews see Ben-Ari et al. 2007; Galanopoulou 2007; Kahle and Staley 2008). However the results of present study of the effect of energy substrates on EGABA and our previous report on modulation of GABA-induced responses by KBs (Rheims et al. 2009) show that EGABA values do not change dramatically during the postnatal period and remain close to Em. Thus, during the early postnatal period [Cl]i is presumably low and GABA has a shunting/inhibitory action. The reasons for this discrepancy can be explained in the light of numerous biochemical studies which demonstrate that energy metabolism in the suckling rodent is largely dependent upon energy substrates other than glucose, e.g. lactate and KBs (Hawkins et al. 1971; Lockwood and Bailey 1971; Page et al. 1971; Cremer and Heath 1974; Yeh and Zee 1976; Pereira de Vasconcelos and Nehlig 1987; Dombrowski et al. 1989; Schroeder et al. 1991; Lust et al. 2003). Glucose utilization alone, at this age, is not efficient enough to meet neuronal energy demands (Nehlig et al. 1988; Dombrowski et al. 1989; Nehlig 1997; Prins 2008) because of the delayed maturation of glycolytic enzymatic system (Land et al. 1977; Leong and Clark 1984a; Dombrowski et al. 1989; Prins 2008). Thus, placing neonatal cortical slices in an environment in which glucose is the sole energy substrate (as is the case in standard ACSF solutions) apparently results in ES deficiency. This leads to an increased [Cl]i and membrane depolarization.

Importantly, energy substrate availability had a prominent effect on the mode of GABAergic synaptic transmission and spontaneous network activity in the cortex. Addition of energy substrates to the extracellular solution induced a strong shift in the mode of GABAergic transmission toward inhibition and suppressed early network oscillations, GDPs. In previous studies, GDPs have been observed in different cortical regions in vitro (Ben-Ari et al. 2007; Allene et al. 2008; Rheims et al. 2008) during postnatal development and are thought to play an important role in the formation of cortical networks (for reviews see Ben-Ari et al. 2007; Ben-Ari 2002; Mohajerani and Cherubini 2006; Sipila and Kaila 2008). The mechanism of GDP generation is still obscure although it is clear that both glutamatergic and GABAergic transmissions are involved in the initiation of GDPs (Ben-Ari 2002; Mohajerani and Cherubini 2006; Ben-Ari et al. 2007; Sipila and Kaila 2008). In our experiments, when energy substrate availability was more adequate, GDPs were strongly inhibited in both neocortex and hippocampus. This suggests that cortical GDPs may represent an oscillatory pattern of network activity with the probability of their generation being directly proportional to energy deficiency. The particular contribution of changes in Em and EGABA to a strong decrease in the probability of GDP generation is as yet unclear. For instance, BHB along with glucose did not prevent GDPs generation while pyruvate did prevent their generation, suggesting that different ES components differently affect spontaneous neuronal activity.

Neuronal depolarization by elevation of [K+]o from 3.5 to 5 mM (a depolarizing shift in K+ current reversal potential of about 10 mV) induced oscillatory network activity in eACSF (Fig. S2). However, an increase in [K+]o may not only cause neuronal depolarization, but also a decrease in K+ currents, alterations in the action potential waveform and other changes in neuronal properties. Importantly, the K+ concentration of 3.5 mM used in both ACSF and eACSF solutions is relevant to the concentration of K+ in interstitial fluid in the brain in vivo (Yamaguchi 1986; Zhang et al. 1990; Silver and Erecinska 1994; Sanchez-Vives and McCormick 2000). In this study, however, we did not investigate the mechanisms of GDP generation in detail.

Our results raise the question of the adequacy of standard ACSF solution(s) (in which glucose is the sole energy substrate) especially for in vitro studies of the immature brain. It is likely that the deficiency in energy substrates in standard ACSF affects not only Em and EGABA, as shown in the present study, but may also adversely affect other energy-dependent neuronal processes. Therefore, we propose a new ACSF solution (eACSF) that more closely resembles the physiological ES pool in vivo.

It is important to note here that we had no intention to reproduce the exact composition of ES pool or exact concentrations of ES as they exist in vivo. Following birth and during early postnatal development, a number of brain parameters, including, for example, the ES pool composition (Vannucci and Duffy 1974; Lust et al. 2003; Erecinska et al. 2004; Prins 2008) and ES transport to neurons by monocarboxylic acid transporters (MCTs) (Rafiki et al. 2003; Vannucci and Simpson 2003; Prins 2008), are dynamically changing each postnatal day. Presumably many enzymatic reactions in slices cannot work as efficiently as in vivo therefore decreasing the neuron ability to utilize energy substrates. We suggest a solution containing a relatively sufficient amount of energy substrates in order to compensate, at least partially, a possible energy deficiency of standard ACSF.

Estimations of glucose and other energy substrate concentrations in the brain’s extracellular fluid vary remarkably. For instance, in different measurements, concentration of glucose ranged from 0.35 to 3.3 mM (McNay and Gold 1999). In suckling rats, normal KB concentrations are about 1–2 mM (Lockwood and Bailey 1971; Page et al. 1971; Yeh and Zee 1976; Ferre et al. 1978; Nehlig and Pereira de Vasconcelos 1993) while lactate concentrations may vary from > 10 mM (newborns) to about 2 mM (Girard et al. 1973; Vannucci and Duffy 1974; Lust et al. 2003). Importantly, both KB and lactate concentrations may strongly depend on physiological conditions (Thurston and Hauhart 1989). Nevertheless, it is unlikely that any ‘overdosage’ of ES in eACSF, contrary to their deficiency, may cause an impairment of cell energy metabolism until ES induce significant acidosis. Indeed, it is well established that the required adjustment in the rate of glycolysis is achieved by a complex interplay among ATP consumption, NADH regeneration, and allosteric regulation of several glycolytic enzymes – including hexokinase, phosphofructokinase-1 (PFK-1), and pyruvate kinase – and by second-to-second fluctuations in the concentration of key metabolites that reflect the cellular balance between ATP production and consumption. ‘The ability of a cell to carry out all these interlocking metabolic processes simultaneously obtaining every product in the amount needed and at the right time, in the face of major perturbations from outside, and without generating leftovers—is an astounding accomplishment. After the protection of its DNA from damage, perhaps nothing is more important to a cell than maintaining a constant supply and concentration of ATP’ (Lehninger 2005).

For instance, high (non-physiological) concentrations of glucose used in standard ACSF (from 10 to 25 mM) in many experiments performed in rodent brain slices were not harmful for neurons. Indeed, the biochemical study revealed that slices utilized in fact less than 3 mM of glucose (Okamoto and Quastel 1970). Complementing glucose with other ES is not a novel approach and has been used previously for different purposes including: systematic utilization of exogenous pyruvate for the survival of neurons in culture; using of both lactate and pyruvate in slice electrophysiology (e.g., Borst et al. 1995; Towers and Hestrin 2008); clinical treatment in cases of e.g. brain injury (Hartmann’s and lactated Ringer’s solutions containing 29 mM of lactate).

In eACSF pyruvate has been substituted for lactate [the energy substrate normally present in the blood (Girard et al. 1973; Nehlig and Pereira de Vasconcelos 1993; Pellerin 2003)]. The reasons for this are: (i) in neurons, lactate is transformed to pyruvate, which is the end product of glycolysis; (ii) pyruvate (as is the case for lactate) readily penetrates the cytoplasmic membrane; (iii) pyruvate is much less expensive than lactate.

The brain’s high energy demands (Shulman et al. 2004; Attwell and Gibb 2005; Korf and Gramsbergen 2007) mean that it is vulnerable to damage induced by metabolic stress (Kunz et al. 2002; Beal 2005; Baron et al. 2007; Sas et al. 2007; de la Torre 2008). To understand the link between neuronal energy supply and abnormal patterns of neuronal network activity, it is necessary to know: which fundamental parameters governing neuronal excitability are particularly sensitive to energy metabolism deficits, and the specific values these parameters have in physiological conditions and in periods of energy deficiency. We suggest that metabolic deficits induce changes in intrinsic neuronal properties resulting in hyperactivity in single neurons and aberrant neuronal network behavior. This hyperactivity in turn increases neuronal energy demands, which cannot be met because of metabolic pathologies and a vicious cycle occurs. Our hypothesis predicts that the adequate delivery of energy substrates may interrupt this pathological spiral of events and provide therapeutic options targeting the cause of pathologies rather than their symptoms.

Footnotes
  • *

    Note, that BHB data shown in Fig. 1 and Fig. 2 are taken from our previous study (Rheims et al. 2009).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Dr. N. Burnashev for discussion of the results and for comments. We thank Dr. T. Zilberter for her valuable help at all stages of this study. We also thank Dr. S. Rheims for his help. This work was supported by a Marie Curie (KBMMGABA No.237327) post-doctoral grant (CH), by the NEUROCYPRES grant from the European Commission Seventh Framework Programme (for MM) and by Institut National de la Santé et de la Recherche Médicale - INSERM (YZ and PB). YZ is the recipient of a Contrat d’interface between INSERM and Centre Hospitalier Universitaire Necker Paris, France. YZ was supported by the MEMOLOAD grant (HEALTH-F2-2007-201159).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Figure S1. eACSF induces outward baseline current. Baseline outward current was induced following exchange of ACSF for eACSF. Gramicidin perforated patch recordings in the same experiment as shown in Fig. 3a. The current trace is filtered at 0.2 Hz low-pass.

Figure S2. Oscillatory network activity induced by increased [K+]O in eACSF. Field recordings in CA3 (P4, mouse). GDPs disappeared following exchange of ACSF with eACSF. However, an increase in [K+]O to 5 mM in eACSF caused the re-appearance of GDP-like network oscillations. The voltage trace was filtered at 0.5 Hz high-pass and 20 Hz low-pass.

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