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Genetically encoded indicators of neuronal activity have diversified and improved in performance in recent years, becoming essential tools for neuroscientists. Lin and Schnitzer review indicators for pH, neurotransmitter, voltage and calcium, with an emphasis on quantifying key indicator attributes and relating them to their applications in neuroscience. Experimental efforts to understand how the brain represents, stores and processes information require high-fidelity recordings of multiple different forms of neural activity within functional circuits. Thus, creating improved technologies for large-scale recordings of neural activity in the live brain is a crucial goal in neuroscience. Over the past two decades, the combination of optical microscopy and genetically encoded fluorescent indicators has become a widespread means of recording neural activity in nonmammalian and mammalian nervous systems, transforming brain research in the process. In this review, we describe and assess different classes of fluorescent protein indicators of neural activity. We first discuss general considerations in optical imaging and then present salient characteristics of representative indicators. Our focus is on how indicator characteristics relate to their use in living animals and on likely areas of future progress.

A new microendoscopic method reveals that hippocampal dendritic spines in the CA1 region undergo a complete turnover in less than six weeks in adult mice; this contrasts with the much greater stability of synapses in the neocortex and provides a physical basis for the fact that episodic memories are only retained by the mouse hippocampus for a few weeks. Dendritic spine turnover in the hippocampus Episodic memories are formed in the mammalian hippocampus but retained there only for a few weeks, compared to the months and years of memory storage provided in the neocortex. Mark Schnitzer and colleagues have used a new microendoscopic method to reveal that hippocampal dendritic spines in the CA1 region undergo complete turnover in less than six weeks in mice, in contrast to the much greater stability of synapses in the neocortex. This work illustrates a possible physical basis for the transience of hippocampal memory processes. The mammalian hippocampus is crucial for episodic memory formation 1 and transiently retains information for about 3–4 weeks in adult mice and longer in humans 2 . Although neuroscientists widely believe that neural synapses are elemental sites of information storage 3 , there has been no direct evidence that hippocampal synapses persist for time intervals commensurate with the duration of hippocampal-dependent memory. Here we tested the prediction that the lifetimes of hippocampal synapses match the longevity of hippocampal memory. By using time-lapse two-photon microendoscopy 4 in the CA1 hippocampal area of live mice, we monitored the turnover dynamics of the pyramidal neurons’ basal dendritic spines, postsynaptic structures whose turnover dynamics are thought to reflect those of excitatory synaptic connections 5 , 6 . Strikingly, CA1 spine turnover dynamics differed sharply from those seen previously in the neocortex 7 , 8 , 9 . Mathematical modelling revealed that the data best matched kinetic models with a single population of spines with a mean lifetime of approximately 1–2 weeks. This implies ∼100% turnover in ∼2–3 times this interval, a near full erasure of the synaptic connectivity pattern. Although N -methyl- d -aspartate (NMDA) receptor blockade stabilizes spines in the neocortex 10 , 11 , in CA1 it transiently increased the rate of spine loss and thus lowered spine density. These results reveal that adult neocortical and hippocampal pyramidal neurons have divergent patterns of spine regulation and quantitatively support the idea that the transience of hippocampal-dependent memory directly reflects the turnover dynamics of hippocampal synapses.

A sizable fraction of granule cells convey information about the expectation of reward, with different populations responding to reward delivery, anticipation and omission, with some responses evolving over time with learning. Reward response in granule cells Classical theories suggest that granule cells in the cerebellum carry sensory and motor signals, enabling downstream Purkinje cells to sense fine contextual changes relating to movement. Using two-photon calcium imaging in behaving mice, Liqun Luo and colleagues also show that a sizable fraction of granule cells convey information about the expectation of reward. Different populations responded to reward delivery, anticipation and omission and some responses evolved over time with learning. The discovery of reward-related signals in granule cells has implications for both models of sensorimotor learning and of cognitive processing in the cerebellum. The human brain contains approximately 60 billion cerebellar granule cells 1 , which outnumber all other brain neurons combined. Classical theories posit that a large, diverse population of granule cells allows for highly detailed representations of sensorimotor context, enabling downstream Purkinje cells to sense fine contextual changes 2 , 3 , 4 , 5 , 6 . Although evidence suggests a role for the cerebellum in cognition 7 , 8 , 9 , 10 , granule cells are known to encode only sensory 11 , 12 , 13 and motor 14 context. Here, using two-photon calcium imaging in behaving mice, we show that granule cells convey information about the expectation of reward. Mice initiated voluntary forelimb movements for delayed sugar-water reward. Some granule cells responded preferentially to reward or reward omission, whereas others selectively encoded reward anticipation. Reward responses were not restricted to forelimb movement, as a Pavlovian task evoked similar responses. Compared to predictable rewards, unexpected rewards elicited markedly different granule cell activity despite identical stimuli and licking responses. In both tasks, reward signals were widespread throughout multiple cerebellar lobules. Tracking the same granule cells over several days of learning revealed that cells with reward-anticipating responses emerged from those that responded at the start of learning to reward delivery, whereas reward-omission responses grew stronger as learning progressed. The discovery of predictive, non-sensorimotor encoding in granule cells is a major departure from the current understanding of these neurons and markedly enriches the contextual information available to postsynaptic Purkinje cells, with important implications for cognitive processing in the cerebellum.

Optical imaging through the intact mouse skull is challenging because of skull-induced aberrations and scattering. We found that three-photon excitation provided improved optical sectioning compared with that obtained with two-photon excitation, even when we used the same excitation wavelength and imaging system. Here we demonstrate three-photon imaging of vasculature through the adult mouse skull at >500-μm depth, as well as GCaMP6s calcium imaging over weeks in cortical layers 2/3 and 4 in awake mice, with 8.5 frames per second and a field of view spanning hundreds of micrometers.

Pain is an unpleasant experience. How the brain’s affective neural circuits attribute this aversive quality to nociceptive information remains unknown. By means of time-lapse in vivo calcium imaging and neural activity manipulation in freely behaving mice encountering noxious stimuli, we identified a distinct neural ensemble in the basolateral amygdala that encodes the negative affective valence of pain. Silencing this nociceptive ensemble alleviated pain affective-motivational behaviors without altering the detection of noxious stimuli, withdrawal reflexes, anxiety, or reward. Following peripheral nerve injury, innocuous stimuli activated this nociceptive ensemble to drive dysfunctional perceptual changes associated with neuropathic pain, including pain aversion to light touch (allodynia). These results identify the amygdalar representations of noxious stimuli that are functionally required for the negative affective qualities of acute and chronic pain perception.

Genetically encoded voltage indicators (GEVIs) are a promising technology for fluorescence readout of millisecond-scale neuronal dynamics. Previous GEVIs had insufficient signaling speed and dynamic range to resolve action potentials in live animals. We coupled fast voltage-sensing domains from a rhodopsin protein to bright fluorophores through resonance energy transfer. The resulting GEVIs are sufficiently bright and fast to report neuronal action potentials and membrane voltage dynamics in awake mice and flies, resolving fast spike trains with 0.2-millisecond timing precision at spike detection error rates orders of magnitude better than previous GEVIs. In vivo imaging revealed sensory-evoked responses, including somatic spiking, dendritic dynamics, and intracellular voltage propagation. These results empower in vivo optical studies of neuronal electrophysiology and coding and motivate further advancements in high-speed microscopy.

The authors use Ca 2+ imaging in freely behaving mice to look at the long-term dynamics of CA1 hippocampal place codes. They find that, in a familiar environment, there is substantial change in the population of place-coding cells over time, but the ensembles of these cells are sufficiently stable to preserve an accurate spatial representation across weeks. Using Ca 2+ imaging in freely behaving mice that repeatedly explored a familiar environment, we tracked thousands of CA1 pyramidal cells' place fields over weeks. Place coding was dynamic, as each day the ensemble representation of this environment involved a unique subset of cells. However, cells in the ∼15–25% overlap between any two of these subsets retained the same place fields, which sufficed to preserve an accurate spatial representation across weeks.

How is it that groups of neurons dispersed through the brain interact to generate complex behaviors? Three papers in this issue present brain-scale studies of neuronal activity and dynamics (see the Perspective by Huk and Hart). Allen et al. found that in thirsty mice, there is widespread neural activity related to stimuli that elicit licking and drinking. Individual neurons encoded task-specific responses, but every brain area contained neurons with different types of response. Optogenetic stimulation of thirst-sensing neurons in one area of the brain reinstated drinking and neuronal activity across the brain that previously signaled thirst. Gründemann et al. investigated the activity of mouse basal amygdala neurons in relation to behavior during different tasks. Two ensembles of neurons showed orthogonal activity during exploratory and nonexploratory behaviors, possibly reflecting different levels of anxiety experienced in these areas. Stringer et al. analyzed spontaneous neuronal firing, finding that neurons in the primary visual cortex encoded both visual information and motor activity related to facial movements. The variability of neuronal responses to visual stimuli in the primary visual area is mainly related to arousal and reflects the encoding of latent behavioral states. Science , this issue p. eaav3932 , p. eaav8736 , p. eaav7893 ; see also p. 236 Longitudinal large-scale imaging of amygdala activity reveals dynamic encoding of behavioral states by two distinct neural ensembles. Internal states, including affective or homeostatic states, are important behavioral motivators. The amygdala regulates motivated behaviors, yet how distinct states are represented in amygdala circuits is unknown. By longitudinally imaging neural calcium dynamics in freely moving mice across different environments, we identified opponent changes in activity levels of two major, nonoverlapping populations of basal amygdala principal neurons. This population signature does not report global anxiety but predicts switches between exploratory and nonexploratory, defensive states. Moreover, the amygdala separately processes external stimuli and internal states and broadcasts state information via several output pathways to larger brain networks. Our findings extend the concept of thalamocortical “brain-state” coding to include affective and exploratory states and provide an entry point into the state dependency of brain function and behavior in defined circuits.

How the brain processes information accurately despite stochastic neural activity is a longstanding question 1 . For instance, perception is fundamentally limited by the information that the brain can extract from the noisy dynamics of sensory neurons. Seminal experiments 2 , 3 suggest that correlated noise in sensory cortical neural ensembles is what limits their coding accuracy 4 – 6 , although how correlated noise affects neural codes remains debated 7 – 11 . Recent theoretical work proposes that how a neural ensemble’s sensory tuning properties relate statistically to its correlated noise patterns is a greater determinant of coding accuracy than is absolute noise strength 12 – 14 . However, without simultaneous recordings from thousands of cortical neurons with shared sensory inputs, it is unknown whether correlated noise limits coding fidelity. Here we present a 16-beam, two-photon microscope to monitor activity across the mouse primary visual cortex, along with analyses to quantify the information conveyed by large neural ensembles. We found that, in the visual cortex, correlated noise constrained signalling for ensembles with 800–1,300 neurons. Several noise components of the ensemble dynamics grew proportionally to the ensemble size and the encoded visual signals, revealing the predicted information-limiting correlations 12 – 14 . Notably, visual signals were perpendicular to the largest noise mode, which therefore did not limit coding fidelity. The information-limiting noise modes were approximately ten times smaller and concordant with mouse visual acuity 15 . Therefore, cortical design principles appear to enhance coding accuracy by restricting around 90% of noise fluctuations to modes that do not limit signalling fidelity, whereas much weaker correlated noise modes inherently bound sensory discrimination. A microscopy system that enables simultaneous recording from hundreds of neurons in the mouse visual cortex reveals that the brain enhances its coding capacity by representing visual inputs in dimensions perpendicular to correlated noise.

Interactions with male and female intruders activated overlapping neuronal populations in the ventromedial hypothalamus of inexperienced adult male mice, and these ensembles gradually separated as the mice acquired social and sexual experience with conspecifics. Social behaviour shapes sex circuitry In the laboratory, most experimentally learned animal behaviours require training to achieve high performance, with neural circuits and ensembles changing as animals master a task. However, it is not clear whether experience can influence circuits for instinctive behaviours, which can be performed without training. Here, David Anderson and colleagues report that modifications to the neural ensembles in the hypothalamus that represent sex occur with increasing social experience. They find that inexperienced animals exhibit overlapping neural representations for male or female members of the same species introduced as intruders to their cage, but sex-specific ensembles emerged over time as mice increased their social and sexual experience. The authors conclude that innate behaviours may not always function as a 'hard-wired' system. All animals possess a repertoire of innate (or instinctive 1 , 2 ) behaviours, which can be performed without training. Whether such behaviours are mediated by anatomically distinct and/or genetically specified neural pathways remains unknown 3 , 4 , 5 . Here we report that neural representations within the mouse hypothalamus, that underlie innate social behaviours, are shaped by social experience. Oestrogen receptor 1-expressing (Esr1 + ) neurons in the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) control mating and fighting in rodents 6 , 7 , 8 . We used microendoscopy 9 to image Esr1 + neuronal activity in the VMHvl of male mice engaged in these social behaviours. In sexually and socially experienced adult males, divergent and characteristic neural ensembles represented male versus female conspecifics. However, in inexperienced adult males, male and female intruders activated overlapping neuronal populations. Sex-specific neuronal ensembles gradually separated as the mice acquired social and sexual experience. In mice permitted to investigate but not to mount or attack conspecifics, ensemble divergence did not occur. However, 30 minutes of sexual experience with a female was sufficient to promote the separation of male and female ensembles and to induce an attack response 24 h later. These observations uncover an unexpected social experience-dependent component to the formation of hypothalamic neural assemblies controlling innate social behaviours. More generally, they reveal plasticity and dynamic coding in an evolutionarily ancient deep subcortical structure that is traditionally viewed as a ‘hard-wired’ system.