Biochemical reactions are sensitive to variations in temperature, pH, O 2 , glucose, etc., of which temperature has been the focus of several studies by clinical neuroscientists1. Temperature fluctuations in the brain on the order of 1–2 °C can impact memory encoding, effect behavioral changes, and generate autonomic responses1. The apparent sensitivity to overall brain temperature originates from reactions at the level of individual neurons. To counteract large external temperature fluctuations, animal cells have evolved certain thermoregulatory mechanisms. For instance, heat shock has been shown to trigger compensatory intracellular endothermic reactions2 that can alter gene expressions and activate signaling cascades3. On the other hand, in adapting to cold environments, for instance, exothermic non-shivering thermogenesis is induced at mitochondria4,5,6 to produce heat. Despite recognition of the fundamental involvement of temperature in eliciting biochemical changes, specific molecular mechanisms for heat evolution in cells are still not clearly identified experimentally7,8,9,10. It is widely known that biological uncoupling proteins (UCP) uncouple oxidative phosphorylation, thereby converting the energy required to synthesize ATP into heat11,12. This steady-state substrate oxidation is expected to produce only ~10−5 K temperature increase per cell7,8,9,10. However, at the onset of proton uncoupling (Fig. 1), a transient proton motive force (pmf) dissipation occurs before enhancing substrate oxidation. In this work, we experimentally demonstrate for the first time that chemically induced pmf dissipation can result in large intracellular temperature spikes of ~4.8 K over a short duration of ~1 s in Aplysia neurons.

Fig. 1 Proton uncoupler in action at the mitochondrial inner membrane. a A schematic of the mitochondrial respiratory chain shows three protein complexes (I, III, IV) producing an H+ gradient across the inner mitochondrial membrane. ATP synthase (AS) utilizes this H+ gradient to synthesize ATP from ADP. b Proton uncouplers allow diffusion of protons through the mitochondrial membrane. This sudden diffusion into the mitochondrial matrix results in a proton current that can generate heat Full size image

Proton uncoupling has been hypothesized to result in transient electrical heating13 (Fig. 1b). The electrochemical proton motive force (Δp) generated by proton pumps (Fig. 1a) are typically 150–200 mV13,14,15. By sharply dissipating the whole mitochondrial potential (~150 mV) using a patch clamp, previous studies16,17 reported an exponentially decreasing proton current (\(I_{{\rm{H}}^ {+}}\)) with a maximum current ~150 pA (Supplementary Fig. 1)16. The resulting heat (\(\dot Q\sim {\mathrm{\Delta }}p \cdot I_{{\rm{H}}^ {+}}\)) can cause a temperature rise in a single mitochondrion at a rate of ~4.8 K/s at the onset of proton motive force dissipation13. This heat pulse is expected to be <1 s owing to the short duration of proton currents (Supplementary Fig. 1)16. We note that the previously reported magnitudes of \(I_{{\rm{H}}^ {+}}\) and Δp, and the duration of proton current may vary across different cell lines depending on the expression of UCP17 and the proton pool. However, irrespective of UCP expression, chemical proton uncouplers can similarly increase the permeability18 of protons across the inner membrane of mitochondria, resulting in a short-lived proton motive force dissipation and associated heating. Previous thermometry on proton uncoupling probed longer temporal scales of ~5 min or more19,20,21,22,23,24 but missed information on short-term pmf dissipation effects. To record transients during pmf dissipation, a thermometry technique that combines low thermal time constant (<1 s) with high accuracy (<±1 K), as well as chemical and electrical inertness, is necessary.

Previous reports of non-invasive thermometry19,22,25 using temperature-dependent fluorescence lifetimes or intensities typically had accuracies26 ≳1 K. They also suffered from off-target signals7,22,27 that came from photobleaching22, variations in microscale viscosity, ion concentrations, and intracellular pH changes within the cellular environment7. Chemically inert micro-fabricated thermocouples28 were previously made that measured extracellular, but not intracellular temperatures. Invasive intracellular thermometers have been made from micropipettes29,30,31,32 and tungsten-based probes33,34. Metal-filled micropipettes31 have a thermal time constant ~0.6 s, which is high for measuring transient pmf dissipation over <1 s. Tungsten probes that have a long (7–10 μm) junction33 measure spatially averaged temperatures inside a cell. Invasive thermometers that are also electrically bare29,30,31,32,33,34 suffer from common mode noise35, when used in an electrically active cellular milieu. Moreover, previous reports29,30,31,32,33,34 typically used a water bath for calibrating the temperature response of the sensors. This can result in errors arising from local convection effects, and temperature differences between reference sensor and the probe. Overall, existing sensing techniques lack the required chemical and electrical inertness, accuracy (<±1 K), and low thermal time constant (<1 s) to measure transient pmf dissipation.

Here, we employed a microscale thermocouple probe to capture such transients in intracellular temperatures. Fig. 2a, b show the probe, fabricated using the techniques of silicon-based microelectromechanical systems (MEMS). Details of the fabrication are explained in Supplementary Fig. 2 and in our previous work26. We performed an on-chip calibration in a vacuum cryostat (Supplementary Fig. 2e), and determined the calibration accuracy to be ~±54 mK at 300 ± 10 K. We note that we do not use a heated culture medium, as was done in previous studies29,30,31,32,33,34, for calibrating the thermal probe. However, we tested our probe in a heated culture medium to confirm the temperature response (Supplementary Fig. 3). The temperature-sensitive Au/Pd thermocouple junction is 1 μm in diameter. It is supported by a 1-μm-thick silicon nitride cantilever tip of 5 μm width. We calculated the time constant of the probe26 to be 32 μs. The probe is electrically insulated with ~300 nm of silicon nitride. In Supplementary Fig. 4, we show that the probe is insulated from common mode signals, resulting in <20 mK noise in a typical electrically active neuron.

Fig. 2 Schematic and microscopy images of intracellular temperature measurement inside Aplysia neurons. a A false-colored scanning electron microscopy image of the thermal probe. The suspended region is ~451-μm long. Scale bar corresponds to 100 μm. b The temperature-sensitive thermocouple junction is ~1 μm in diameter. Scale bar corresponds to 5 μm. c A schematic of the setup used for measuring temperature changes inside the cell while concurrently monitoring the membrane potential using a KCl sharp microelectrode. The brown patches in the perinuclear cytoplasm are representative of mitochondrial sites in Aplysia neurons38. d An optical image of the abdominal ganglion of Aplysia. The two probes are inside the target cell R15. Scale bar corresponds to 100 μm Full size image

We made intracellular measurements on neurons from the sea slug Aplysia californica. The animal’s abdominal ganglion, which constitutes parts of a distributed central nervous system, possesses neurons that can reach up to ~1 mm in diameter36, with nuclei37 as large as ~800 μm. The perinuclear cytoplasm is enriched with mitochondria38,39. The Aplysia neurons found superficially in the abdominal ganglion are typically hundreds of microns in diameter, which renders them favorable to penetration with our thermal probe that is ~5-μm wide. Unless mentioned otherwise, we used neurons from the abdominal ganglia throughout the study. We also utilized a sharp voltage microelectrode to record the real-time membrane potential of the neuron as a metric of cell health (Fig. 2c). More details on the microelectrode and culture dish preparation are in the Methods section. Figure 2d shows an optical microscope image of the culture dish with thermal probe and voltage microelectrode inside a neuron.

In this work, we measure transient intracellular temperature changes during proton motive force dissipation, which is induced by chemical proton uncouplers. We first identify off-target responses to the proton uncouplers. Then, we extract intracellular temperature responses from proton motive force dissipation.