Pupil diameter is often treated as a noninvasive readout of activity in the locus coeruleus (LC). However, how accurately it can be used to index LC activity is not known. To address this question, we established a graded relationship between pupil size changes and LC spiking activity in mice, where pupil dilation increased monotonically with the number of LC spikes. However, this relationship exists with substantial variability such that pupil diameter can only be used to accurately predict a small fraction of LC activity on a moment-by-moment basis. In addition, pupil exhibited large session-to-session fluctuations in response to identical optical stimulation in the LC. The variations in the pupil–LC relationship were strongly correlated with decision bias-related behavioral variables. Together, our data show that substantial variability exists in an overall graded relationship between pupil diameter and LC activity, and further suggest that the pupil–LC relationship is dynamically modulated by brain states, supporting and extending our previous findings (Yang et al., 2021).

Neurons in the locus coeruleus (LC) have been traditionally viewed as a homogenous population. Recent studies are beginning to uncover heterogeneous molecular phenotypes and projection targets in this nucleus, but how such heterogeneity is ultimately manifested in neuronal physiology and function is largely unknown. We took an orthogonal approach to directly assess the physiological and functional heterogeneity in the LC. By identifying noradrenergic neurons using a genetic-based tagging approach, our study revealed a subset of neurons exhibiting a distinctive narrow spike waveform and lacking the prolonged after-hyperpolarization. We further provided evidence establishing a link between the proximity of LC neurons and their response latency, where putatively proximal neurons tended to have a long latency to optogenetic activation. Finally, we found that response latency of LC neurons was correlated with their burst firing and distinct relationship with pupil diameter. Together, our study presents novel evidence to reveal and link LC heterogeneity at the physiological level to that at the functional level.

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