Energetic substrate availability regulates synchronous activity in an excitatory neural network

Abstract

Neural networks are required to meet significant metabolic demands associated with performing sophisticated computational tasks in the brain. The necessity for efficient transmission of information imposes stringent constraints on the metabolic pathways that can be used for energy generation at the synapse, and thus low availability of energetic substrates can reduce the efficacy of synaptic function. Here we study the effects of energetic substrate availability on global neural network behavior and find that glucose alone can sustain excitatory neurotransmission required to generate high-frequency synchronous bursting that emerges in culture. In contrast, obligatory oxidative energetic substrates such as lactate and pyruvate are unable to substitute for glucose, indicating that processes involving glucose metabolism form the primary energy-generating pathways supporting coordinated network activity. Our experimental results are discussed in the context of the role that metabolism plays in supporting the performance of individual synapses, including the relative contributions from postsynaptic responses, astrocytes, and presynaptic vesicle cycling. We propose a simple computational model for our excitatory cultures that accurately captures the inability of metabolically compromised synapses to sustain synchronous bursting when extracellular glucose is depleted.

Fig 1. Synchronous bursting characteristics.

A) Synchronized bursts consisting of trains of action potentials are clearly visible in raw electrode data. Representative data from two neighboring electrodes showing temporal correlation of bursts occurring in control conditions (i). Synchronized bursts are obliterated (although spontaneous action potentials persist) by the presence of the AMPA/kainite receptor antagonist CNQX (ii), but this effect is immediately reversible following a wash-off (iii). Total inhibition of electrical activity upon treatment with TTX (iv). For illustrative purposes, upper panels displaying fast time scale are smoothed using a 2ms Gaussian window. B) Numbers of synchronous bursts per minute (SBPM) gradually increases and stabilizes over the course of network development (data from three independent 6-week-long experiments each using distinct hESC clones and astrocyte preparations). Two-way ANOVA reports time (days from induction) as the major source of variation, P_time < 0.0001; N = 3. C) When mature, the same cultures were subject to CNQX treatment, revealing the dependence of synchronous bursting on excitatory glutamatergic signaling. Zero synchronous bursts were observed in the presence of CNQX.

Fig 2. Inhibiting the vesicle recycling and maintenance pathways.

A) Incubating cultures in the presence of 50nM CMA for 10min significantly reduces the frequency of synchronous bursting compared to 10min incubation in the presence of vehicle alone. CMA versus vehicle P = 0.032; N = 3. B) 80μM dynasore had no detectible effect on SBPM after the first, or second, 10min incubation. Dynasore 10min versus vehicle P = 0.4, dynasore 20min versus vehicle P = 0.48; N = 3.

Fig 3. Glucose depletion decreases SBPM.

A) Cartoon schematic of substrate depletion-repletion experimental protocol described in Materials and Methods. Each wash-incubate-equilibrate-record epoch was performed using fresh media regardless of substrate composition. B) Synchronous burst frequency decreases following 10min incubation in the absence of extracellular glucose (25mM) and pyruvate (0.22mM), and significantly further still following a second 10min incubation. Replenishment of glucose alone is sufficient to restore a significantly higher SBPM. No substrate 20min versus glucose and pyruvate P = 0.013; no substrate 20min versus no substrate 10min P = 0.003; glucose alone versus no substrate 20min P = 0.012; N = 4. C) Only the metabolically active D-isoform of glucose (not L-glucose) can sustain a significantly higher SBPM in physiologically-relevant conditions containing 5mM D- or L-glucose, 5mM DL-lactate (racemic mixture) and 0.22mM pyruvate. L-glucose 20min versus D-glucose control P < 0.001; L-glucose 20min versus L-glucose 10min P = 0.027; D-glucose replenishment versus L-glucose 20min P = 0.014; N = 4.

Fig 4. Computational model of synchronous bursting.

A) Cartoon schematic showing that presynaptic vesicle recycling involves several steps suggested to depend on ATP supplied by glycolysis, including endocytosis and vesicle re-acidification by the v-ATPase. In our computational model we encompassed contributions from all pathways in a single term describing the rate of synaptic vesicle recovery and maintenance. B) Simulations reveal that in high levels of extracellular glucose (left), synchronized bursting persists at a higher frequency than when extracellular glucose is low (right), as modelled by reducing the rate of vesicle recovery and maintenance. Upper panels display raster plots of spike timings from all 400 neurons in the simulated network, middle panels the total spike count across the network as a function of time, and lower panels the corresponding fluctuations in membrane potential of a representative neuron.