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).
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.
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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.
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.
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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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