Differential Control of Inhibitory and Excitatory Nerve Terminal Function by Mitochondria

Abstract

Inhibitory neurons shape the brain's computational landscape and rely on different cellular architectures and intrinsic properties than excitatory neurons. Maintenance of the overall balance of excitatory (E) versus inhibitory (I) drive is essential, as disruptions can lead to neuropathological conditions, including autism and epilepsy. Metabolic perturbations are a common driver of E/I imbalance but differential sensitivity of these two neuron types to metabolic lesions is not well understood. Here, we characterized differences in presynaptic bioenergetic regulation between excitatory and inhibitory nerve terminals using genetically encoded indicators expressed in primary dissociated neuronal cultures. Our experiments showed that inhibitory nerve terminals sustain higher ATP levels than excitatory nerve terminals arising from increased mitochondrial metabolism. Additionally, mitochondria in inhibitory neurons play a greater role in buffering presynaptic Ca2+ and inhibitory mitochondrial Ca2+ handling is differentially regulated by TMEM65-meditaed acceleration of mitochondrial Ca2+ extrusion following bursts of activity. These experiments thus identify differential reliance on mitochondrial function across two major neuron types.

Characterization of β-Hydroxybutyrate as a Cell Autonomous Fuel for Active Excitatory and Inhibitory Neurons

“…Given the similarity of resting ATP levels between neurons in BHB and neurons in lactate/pyruvate ( Figure 2A , Supplementary Figure 2A ), we examined iATPSnFR2.0-HALO fluorescence recovery following a burst of AP firing capable of transiently depressing presynaptic ATP levels, for neurons bathed in either BHB or lactate/pyruvate. Following a 60 second burst of 10 Hz AP firing, presynaptic ATP recovered much more slowly in excitatory compared to inhibitory neurons when fueled by lactate/pyruvate ( Figure 2C – E ), as previously reported ( Bredvik and Ryan, 2024 ). Inhibitory neurons in BHB recovered much more slowly than inhibitory neurons in lactate/pyruvate ( Figure 2B – 2D ).…”

“…One notable finding is that under pure BHB conditions, inhibitory neurons sustain much higher steady-state presynaptic ATP levels than excitatory neurons (Figure 2A), consistent with our previous observation that there is a higher abundance of mitochondria in inhibitory neurons (Bredvik and Ryan, 2024). We found that although both subtypes could cell autonomously use BHB as a fuel for synaptic transmission, in the stimulus paradigms examined (10 Hz firing in bursts at minute intervals or sustained for 60 seconds) inhibitory neurons performed no better than excitatory neurons when provided BHB as fuel (Figure 1, Figure 3, Supplementary Figure 1, Supplementary Figure 2).…”

“…To directly compare the metabolic state of inhibitory and excitatory neurons using mitochondrial fuel sources, we targeted the recently improved ratiometric ATP sensor iATPSnFR2.0-HALO (Marvin et al, 2024) to the cytosol of either excitatory or inhibitory neurons using CaMKII and hDlxI56i specific expression constructs. We recently showed that, in agreement with in vivo findings (Nie and Wong-Riley, 1995;Gulyás et al, 2006), cultured inhibitory axons have a ~25% higher density of mitochondria compared to excitatory axons, resulting in much higher resting ATP levels when axons are fueled solely by mitochondrial metabolism using a mixture of lactate and pyruvate (Bredvik and Ryan, 2024). Following 5 minutes of perfusion in BHB, we found that resting ATP levels were ~3-fold higher in inhibitory compared to excitatory axons (Figure 2A), consistent with the notion that neuronal mitochondria carry out ketolysis (Figure 1) and that inhibitory neurons have a greater total mitochondrial capacity than excitatory neurons.…”

“…Fluorescent report constructs CaMKII synaptophysin-pHluorin, hDlxI56i synaptophysin-pHluorin, hDlxI56i iATPSnFR2.0-HALO ( Bredvik and Ryan, 2024 ) and CaMKII iATPSnFR2.0-HALO ( Marvin et al, 2024 ) are available on Addgene.…”