Fast and slow: Recording neuromodulator dynamics across both transient and chronic time scales.

Neuromodulators transform animal behaviors. Recent research has demonstrated the importance of both sustained and transient change in neuromodulators, likely due to tonic and phasic neuromodulator release. However, no method could simultaneously record both types of dynamics. Fluorescence lifetime of optical reporters could offer a solution because it allows high temporal resolution and is impervious to sensor expression differences across chronic periods. Nevertheless, no fluorescence lifetime change across the entire classes of neuromodulator sensors was previously known. Unexpectedly, we find that several intensity-based neuromodulator sensors also exhibit fluorescence lifetime responses. Furthermore, we show that lifetime measures in vivo neuromodulator dynamics both with high temporal resolution and with consistency across animals and time. Thus, we report a method that can simultaneously measure neuromodulator change over transient and chronic time scales, promising to reveal the roles of multi-time scale neuromodulator dynamics in diseases, in response to therapies, and across development and aging.

A Cre-dependent reporter mouse for quantitative real-time imaging of protein kinase A activity dynamics.

Intracellular signaling dynamics play a crucial role in cell function. Protein kinase A (PKA) is a key signaling molecule that has diverse functions, from regulating metabolism and brain activity to guiding development and cancer progression. We previously developed an optical reporter, FLIM-AKAR, that allows for quantitative imaging of PKA activity via fluorescence lifetime imaging microscopy and photometry. However, using viral infection or electroporation for the delivery of FLIM-AKAR is invasive and results in variable expression. Here, we developed a reporter mouse, FL-AK, which expresses FLIM-AKAR in a Cre-dependent manner from the ROSA26 locus. FL-AK provides robust and consistent expression of FLIM-AKAR over time. Functionally, the mouse line reports an increase in PKA activity in response to activation of both Gαs and Gαq-coupled receptors in brain slices. In vivo, FL-AK reports PKA phosphorylation in response to neuromodulator receptor activation. Thus, FL-AK provides a quantitative, robust, and flexible method to reveal the dynamics of PKA activity in diverse cell types.

A Cre-dependent reporter mouse for quantitative real-time imaging of Protein Kinase A activity dynamics.

Intracellular signaling dynamics play a crucial role in cell function. Protein kinase A (PKA) is a key signaling molecule that has diverse functions, from regulating metabolism and brain activity to guiding development and cancer progression. We previously developed an optical reporter, FLIM-AKAR, that allows for quantitative imaging of PKA activity via fluorescence lifetime imaging microscopy and photometry. However, using viral infection or electroporation for the delivery of FLIM-AKAR is invasive, cannot easily target sparse or hard-to-transfect/infect cell types, and results in variable expression. Here, we developed a reporter mouse, FL-AK, which expresses FLIM-AKAR in a Cre-dependent manner from the ROSA26 locus. FL-AK provides robust and consistent expression of FLIM-AKAR over time. Functionally, the mouse line reports an increase in PKA activity in response to activation of both Gαs and Gαq-coupled receptors in brain slices. In vivo, FL-AK reports PKA phosphorylation in response to neuromodulator receptor activation. Thus, FL-AK provides a quantitative, robust, and flexible method to reveal the dynamics of PKA activity in diverse cell types.

Rapid and active stabilization of visual cortical firing rates across light-dark transitions.

The dynamics of neuronal firing during natural vision are poorly understood. Surprisingly, mean firing rates of neurons in primary visual cortex (V1) of freely behaving rodents are similar during prolonged periods of light and darkness, but it is unknown whether this reflects a slow adaptation to changes in natural visual input or insensitivity to rapid changes in visual drive. Here, we use chronic electrophysiology in freely behaving rats to follow individual V1 neurons across many dark-light (D-L) and light-dark (L-D) transitions. We show that, even on rapid timescales (1 s to 10 min), neuronal activity was only weakly modulated by transitions that coincided with the expected 12-/12-h L-D cycle. In contrast, a larger subset of V1 neurons consistently responded to unexpected L-D and D-L transitions, and disruption of the regular L-D cycle with 60 h of complete darkness induced a robust increase in V1 firing on reintroduction of visual input. Thus, V1 neurons fire at similar rates in the presence or absence of natural stimuli, and significant changes in activity arise only transiently in response to unexpected changes in the visual environment. Furthermore, although mean rates were similar in light and darkness, pairwise correlations were significantly stronger during natural vision, suggesting that information about natural scenes in V1 may be more strongly reflected in correlations than individual firing rates. Together, our findings show that V1 firing rates are rapidly and actively stabilized during expected changes in visual input and are remarkably stable at both short and long timescales.