Simultaneous electrophysiology and optogenetic perturbation of the same neurons in chronically implanted animals using µLED silicon probes.

Micro-light-emitting-diode (µLED) silicon probes feature independently controllable miniature light-emitting-diodes (LEDs) embedded at several positions in each shank of a multi-shank probe, enabling temporally and spatially precise optogenetic neural circuit interrogation. Here, we present a protocol for performing causal and reproducible neural circuit manipulations in chronically implanted, freely moving animals. We describe steps for introducing optogenetic constructs, preparing and implanting a µLED probe, performing simultaneous in vivo electrophysiology with focal optogenetic perturbation, and recovering a probe following termination of an experiment. For complete details on the use and execution of this protocol, please refer to Watkins de Jong et al. (2023).1.

Erasable Hippocampal Neural Signatures Predict Memory Discrimination.

Memories involving the hippocampus can take several days to consolidate, challenging efforts to uncover the neuronal signatures underlying this process. Using calcium imaging in freely moving mice, we tracked the hippocampal dynamics underlying memory formation across a ten-day contextual fear conditioning (CFC) task. We found that cell turnover between the conditioning chamber and a neutral arena even prior to learning predicted the accuracy of subsequent memory recall the next day. Following learning, context-specific place field remapping correlated with memory performance. To causally test whether these hippocampal dynamics support memory consolidation, we induced amnesia in a group of mice by pharmacologically blocking protein synthesis immediately following learning. We found that halting protein synthesis following learning paradoxically accelerated cell turnover and also arrested learning-related remapping, paralleling the absence of remapping observed in untreated mice that exhibited poor memory expression. Finally, coordinated neural activity that emerged following learning was dependent on intact protein synthesis and predicted memory-related freezing behavior. We conclude that context-specific place field remapping and the development of coordinated ensemble activity require protein synthesis and underlie contextual fear memory consolidation.

Simultaneous Electrophysiology and Optogenetic Perturbation of the Same Neurons in Chronically Implanted Animals using µLED Silicon Probes.

Optogenetics are a powerful tool for testing how a neural circuit influences neural activity, cognition, and behavior. Accordingly, the number of studies employing optogenetic perturbation has grown exponentially over the last decade. However, recent studies have highlighted that the impact of optogenetic stimulation/silencing can vary depending on the construct used, the local microcircuit connectivity, extent/power of illumination, and neuron types perturbed. Despite these caveats, the majority of studies employ optogenetics without simultaneously recording neural activity in the circuit that is being perturbed. This dearth of simultaneously recorded neural data is due in part to technical difficulties in combining optogenetics and extracellular electrophysiology. The recent introduction of µLED silicon probes, which feature independently controllable miniature LEDs embedded at several levels of each of multiple shanks of silicon probes, provides a tractable method for temporally and spatially precise interrogation of neural circuits. Here, we provide a protocol addressing how to perform chronic recordings using µLED probes. This protocol provides a schematic for performing causal and reproducible interrogations of neural circuits and addresses all phases of the recording process: introduction of optogenetic construct, implantation of the µLED probe, performing simultaneous optogenetics and electrophysiology in vivo , and post-processing of recorded data. SUMMARY: This method allows a researcher to simultaneously perturb neural activity and record electrophysiological signal from the same neurons with high spatial specificity using silicon probes with integrated µLEDs. We outline a procedure detailing all stages of the process for performing reliable µLED experiments in chronically implanted rodents.

Hippocampal spatial memory representations in mice are heterogeneously stable.

The population of hippocampal neurons actively coding space continually changes across days as mice repeatedly perform tasks. Many hippocampal place cells become inactive while other previously silent neurons become active, challenging the idea that stable behaviors and memory representations are supported by stable patterns of neural activity. Active cell replacement may disambiguate unique episodes that contain overlapping memory cues, and could contribute to reorganization of memory representations. How active cell replacement affects the evolution of representations of different behaviors within a single task is unknown. We trained mice to perform a delayed nonmatching to place task over multiple weeks, and performed calcium imaging in area CA1 of the dorsal hippocampus using head-mounted miniature microscopes. Cells active on the central stem of the maze "split" their calcium activity according to the animal's upcoming turn direction (left or right), the current task phase (study or test), or both task dimensions, even while spatial cues remained unchanged. We found that, among reliably active cells, different splitter neuron populations were replaced at unequal rates, resulting in an increasing number of cells modulated by turn direction and a decreasing number of cells with combined modulation by both turn direction and task phase. Despite continual reorganization, the ensemble code stably segregated these task dimensions. These results show that hippocampal memories can heterogeneously reorganize even while behavior is unchanging.

Neurophysiological coding of space and time in the hippocampus, entorhinal cortex, and retrosplenial cortex.

Neurophysiological recordings in behaving rodents demonstrate neuronal response properties that may code space and time for episodic memory and goal-directed behaviour. Here, we review recordings from hippocampus, entorhinal cortex, and retrosplenial cortex to address the problem of how neurons encode multiple overlapping spatiotemporal trajectories and disambiguate these for accurate memory-guided behaviour. The solution could involve neurons in the entorhinal cortex and hippocampus that show mixed selectivity, coding both time and location. Some grid cells and place cells that code space also respond selectively as time cells, allowing differentiation of time intervals when a rat runs in the same location during a delay period. Cells in these regions also develop new representations that differentially code the context of prior or future behaviour allowing disambiguation of overlapping trajectories. Spiking activity is also modulated by running speed and head direction, supporting the coding of episodic memory not as a series of snapshots but as a trajectory that can also be distinguished on the basis of speed and direction. Recent data also address the mechanisms by which sensory input could distinguish different spatial locations. Changes in firing rate reflect running speed on long but not short time intervals, and few cells code movement direction, arguing against path integration for coding location. Instead, new evidence for neural coding of environmental boundaries in egocentric coordinates fits with a modelling framework in which egocentric coding of barriers combined with head direction generates distinct allocentric coding of location. The egocentric input can be used both for coding the location of spatiotemporal trajectories and for retrieving specific viewpoints of the environment. Overall, these different patterns of neural activity can be used for encoding and disambiguation of prior episodic spatiotemporal trajectories or for planning of future goal-directed spatiotemporal trajectories.

Trajectory-modulated hippocampal neurons persist throughout memory-guided navigation.

Trajectory-dependent splitter neurons in the hippocampus encode information about a rodent's prior trajectory during performance of a continuous alternation task. As such, they provide valuable information for supporting memory-guided behavior. Here, we employed single-photon calcium imaging in freely moving mice to investigate the emergence and fate of trajectory-dependent activity through learning and mastery of a continuous spatial alternation task. In agreement with others, the quality of trajectory-dependent information in hippocampal neurons correlated with task performance. We thus hypothesized that, due to their utility, splitter neurons would exhibit heightened stability. We find that splitter neurons were more likely to remain active and retained more consistent spatial information across multiple days than other neurons. Furthermore, we find that both splitter neurons and place cells emerged rapidly and maintained stable trajectory-dependent/spatial activity thereafter. Our results suggest that neurons with useful functional coding exhibit heightened stability to support memory guided behavior.

Hippocampal Place Fields Maintain a Coherent and Flexible Map across Long Timescales.

To provide a substrate for remembering where in space events have occurred, place cells must reliably encode the same positions across long timescales. However, in many cases, place cells exhibit instability by randomly reorganizing their place fields between experiences, challenging this premise. Recent evidence suggests that, in some cases, instability could also arise from coherent rotations of place fields, as well as from random reorganization. To investigate this possibility, we performed in vivo calcium imaging in dorsal hippocampal region CA1 of freely moving mice while they explored two arenas with different geometry and visual cues across 8 days. The two arenas were rotated randomly between sessions and then connected, allowing us to probe how cue rotations, the integration of new information about the environment, and the passage of time concurrently influenced the spatial coherence of place fields. We found that spatially coherent rotations of place-field maps in the same arena predominated, persisting up to 6 days later, and that they frequently rotated in a manner that did not match that of the arena rotation. Furthermore, place-field maps were flexible, as mice frequently employed a similar, coherent configuration of place fields to represent each arena despite their differing geometry and eventual connection. These results highlight the ability of the hippocampus to retain consistent relationships between cells across long timescales and suggest that, in many cases, apparent instability might result from a coherent rotation of place fields.

The Same Hippocampal CA1 Population Simultaneously Codes Temporal Information over Multiple Timescales.

It has long been hypothesized that a primary function of the hippocampus is to discover and exploit temporal relationships between events. Previously, it has been reported that sequences of "time cells" in the hippocampus extend for tens of seconds. Other studies have shown that neuronal firing in the hippocampus fluctuates over hours and days. Both of these mechanisms could enable temporal encoding of events over very different timescales. However, thus far, these two classes of phenomena have never been observed simultaneously, which is necessary to ascribe broad-range temporal coding to the hippocampus. Using in vivo calcium imaging in unrestrained mice, we observed sequences of hippocampal neurons that bridged a 10 s delay. Similar sequences were observed over multiple days, but the set of neurons participating in those sequences changed gradually. Thus, the same population of neurons that encodes temporal information over seconds can also be used to distinguish periods of time over much longer timescales. These results unify two previously separate paradigms of temporal processing in the hippocampus that support episodic memory.

Hippocampal representation of related and opposing memories develop within distinct, hierarchically organized neural schemas.

Recent evidence suggests that the hippocampus may integrate overlapping memories into relational representations, or schemas, that link indirectly related events and support flexible memory expression. Here we explored the nature of hippocampal neural population representations for multiple features of events and the locations and contexts in which they occurred. Hippocampal networks developed hierarchical organizations of associated elements of related but separately acquired memories within a context, and distinct organizations for memories where the contexts differentiated object-reward associations. These findings reveal neural mechanisms for the development and organization of relational representations.