Hypoxia tolerance in the vertebrate brain often involves chemical modulators that arrest neuronal activity to conserve energy. However, in intact networks, it can be difficult to determine whether hypoxia triggers modulators to stop activity in a protective manner or whether activity stops because rates of ATP synthesis are insufficient to support network function. Here, we assessed the extent to which neuromodulation or metabolic limitations arrest activity in the respiratory network of bullfrogs-a circuit that survives moderate periods of oxygen deprivation, presumably, by activating an inhibitory noradrenergic pathway. We confirmed that hypoxia and norepinephrine (NE) reduce network output, consistent with the view that hypoxia may cause the release of NE to inhibit activity. However, these responses differed qualitatively; hypoxia, but not NE, elicited a large motor burst and silenced the network. The stereotyped response to hypoxia persisted in the presence of both NE and an adrenergic receptor blocker that eliminates sensitivity to NE, indicating that noradrenergic signaling does not cause the arrest. Pharmacological inhibition of glycolysis and mitochondrial respiration recapitulated all features of hypoxia, implying that reduced ATP synthesis underlies the effects of hypoxia on network activity. Finally, activating modulatory mechanisms that dampen neuronal excitability when ATP falls, KATP channels and AMP-dependent protein kinase, did not resemble the hypoxic response. These results suggest energy failure- rather than inhibitory modulation- silences the respiratory network during hypoxia and emphasize the need to account for metabolic limitations before concluding that modulators arrest activity as an adaptation for energy conservation in the nervous system.
Neural networks tune synaptic and cellular properties to produce stable activity. One form of homeostatic regulation involves scaling the strength of synapses up or down in a global and multiplicative manner to oppose activity disturbances. In American bullfrogs, excitatory synapses scale up to regulate breathing motor function after inactivity in hibernation, connecting homeostatic compensation to motor behavior. In traditional models of homeostatic synaptic plasticity, inactivity is thought to increase synaptic strength via mechanisms that involve reduced Ca2+ influx through voltage-gated channels. Therefore, we tested whether pharmacological inactivity and inhibition of voltage-gated Ca2+ channels are sufficient to drive synaptic compensation in this system. For this, we chronically exposed ex vivo brainstem preparations containing the intact respiratory network to tetrodotoxin (TTX) to stop activity and nimodipine to block L-type Ca2+ channels. We show that hibernation and TTX similarly increased motoneuron synaptic strength and that hibernation occluded the response to TTX. In contrast, inhibiting L-type Ca2+ channels did not upregulate synaptic strength but disrupted the apparent multiplicative scaling of synaptic compensation typically observed in response to hibernation. Thus, inactivity drives up synaptic strength through mechanisms that do not rely on reduced L-type channel function, while Ca2+ signaling associated with the hibernation environment independently regulates the balance of synaptic weights. Altogether, these results point to multiple feedback signals for shaping synaptic compensation that gives rise to proper network function during environmental challenges in vivo.
Synaptic scaling is a compensation mechanism that adjusts all synapses by the same relative amount to regulate neuronal activity. However, synaptic compensation does not always scale uniformly, leaving to question if a scaling rule is required to regulate circuit output. We previously showed that scaling up excitatory synapses on motoneurons regulates the respiratory network following inactivity caused by hibernation in frogs (Santin et al., 2017). Although synaptic scaling is thought to involve a scaling factor that multiplicatively upregulates synaptic strength, we find here that distinct mechanisms account for the upregulation of mean synaptic strength and the scaling organization. These processes are separable, as blocking L-type Ca2+ channels undoes the scaling pattern of compensation but does not interfere with the mean increase in synaptic drive. Strikingly, motor output from the respiratory network was weaker when motoneuron synapses were "unscaled" despite having the same average amount of compensation after hibernation. Thus, parallel mechanisms regulate the amount and organizational pattern of synaptic scaling, and both must work appropriately for proper network function. Collectively, these results emphasize that an apparently normal amount of synaptic compensation may still lead to circuit dysfunction in neurological disorders if the balance of synaptic inputs is not accurately regulated.
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