Lateral entorhinal cortex subpopulations represent experiential epochs surrounding reward.

During goal-directed navigation, 'what' information, describing the experiences occurring in periods surrounding a reward, can be combined with spatial 'where' information to guide behavior and form episodic memories. This integrative process likely occurs in the hippocampus, which receives spatial information from the medial entorhinal cortex; however, the source of the 'what' information is largely unknown. Here, we show that mouse lateral entorhinal cortex (LEC) represents key experiential epochs during reward-based navigation tasks. We discover separate populations of neurons that signal goal approach and goal departure and a third population signaling reward consumption. When reward location is moved, these populations immediately shift their respective representations of each experiential epoch relative to reward, while optogenetic inhibition of LEC disrupts learning the new reward location. Therefore, the LEC contains a stable code of experiential epochs surrounding and including reward consumption, providing reward-centric information to contextualize the spatial information carried by the medial entorhinal cortex.

A robot-rodent interaction arena with adjustable spatial complexity for ethologically relevant behavioral studies.

Outside of the laboratory, animals behave in spaces where they can transition between open areas and coverage as they interact with others. Replicating these conditions in the laboratory can be difficult to control and record. This has led to a dominance of relatively simple, static behavioral paradigms that reduce the ethological relevance of behaviors and may alter the engagement of cognitive processes such as planning and decision-making. Therefore, we developed a method for controllable, repeatable interactions with others in a reconfigurable space. Mice navigate a large honeycomb lattice of adjustable obstacles as they interact with an autonomous robot coupled to their actions. We illustrate the system using the robot as a pseudo-predator, delivering airpuffs to the mice. The combination of obstacles and a mobile threat elicits a diverse set of behaviors, such as increased path diversity, peeking, and baiting, providing a method to explore ethologically relevant behaviors in the laboratory.

Full field-of-view virtual reality goggles for mice.

Visual virtual reality (VR) systems for head-fixed mice offer advantages over real-world studies for investigating the neural circuitry underlying behavior. However, current VR approaches do not fully cover the visual field of view of mice, do not stereoscopically illuminate the binocular zone, and leave the lab frame visible. To overcome these limitations, we developed iMRSIV (Miniature Rodent Stereo Illumination VR)-VR goggles for mice. Our system is compact, separately illuminates each eye for stereo vision, and provides each eye with an ∼180° field of view, thus excluding the lab frame while accommodating saccades. Mice using iMRSIV while navigating engaged in virtual behaviors more quickly than in a current monitor-based system and displayed freezing and fleeing reactions to overhead looming stimulation. Using iMRSIV with two-photon functional imaging, we found large populations of hippocampal place cells during virtual navigation, global remapping during environment changes, and unique responses of place cell ensembles to overhead looming stimulation.

Lateral entorhinal cortex subpopulations represent experiential epochs surrounding reward.

During goal-directed navigation, "what" information, which describes the experiences occurring in periods surrounding a reward, can be combined with spatial "where" information to guide behavior and form episodic memories1,2. This integrative process is thought to occur in the hippocampus3, which receives spatial information from the medial entorhinal cortex (MEC)4; however, the source of the "what" information and how it is represented is largely unknown. Here, by establishing a novel imaging method, we show that the lateral entorhinal cortex (LEC) of mice represents key experiential epochs during a reward-based navigation task. We discover a population of neurons that signals goal approach and a separate population of neurons that signals goal departure. A third population of neurons signals reward consumption. When reward location is moved, these populations immediately shift their respective representations of each experiential epoch relative to reward, while optogenetic inhibition of LEC disrupts learning of the new reward location. Together, these results indicate the LEC provides a stable code of experiential epochs surrounding and including reward consumption, providing reward-centric information to contextualize the spatial information carried by the MEC. Such parallel representations are well-suited for generating episodic memories of rewarding experiences and guiding flexible and efficient goal-directed navigation5-7.

Unique functional responses differentially map onto genetic subtypes of dopamine neurons.

Dopamine neurons are characterized by their response to unexpected rewards, but they also fire during movement and aversive stimuli. Dopamine neuron diversity has been observed based on molecular expression profiles; however, whether different functions map onto such genetic subtypes remains unclear. In this study, we established that three genetic dopamine neuron subtypes within the substantia nigra pars compacta, characterized by the expression of Slc17a6 (Vglut2), Calb1 and Anxa1, each have a unique set of responses to rewards, aversive stimuli and accelerations and decelerations, and these signaling patterns are highly correlated between somas and axons within subtypes. Remarkably, reward responses were almost entirely absent in the Anxa1+ subtype, which instead displayed acceleration-correlated signaling. Our findings establish a connection between functional and genetic dopamine neuron subtypes and demonstrate that molecular expression patterns can serve as a common framework to dissect dopaminergic functions.

Molecular heterogeneity in the substantia nigra: A roadmap for understanding PD motor pathophysiology.

As the ability to capture single-cell expression profiles has grown in recent years, neuroscientists studying a wide gamut of brain regions have discovered remarkable heterogeneity within seemingly related populations (Saunders et al., 2018a; Zeisel et al., 2015). These "molecular subtypes" have been demonstrated even within brain nuclei expressing the same neurotransmitter (Saunders et al., 2018a; Poulin et al., 2020; Ren et al., 2019; Okaty et al., 2020). Recently, dopamine (DA) neurons of the substantia nigra pars compacta (SNc) and adjacent ventral tegmental area (VTA) have been revealed to be diverse not only when comparing between these two dopaminergic nuclei, but within them, and with the distribution of identified subtypes often agnostic to traditional neuroanatomical boundaries (Saunders et al., 2018a; Hook et al., 2018; Kramer et al., 2018; La Manno et al., 2016; Poulin et al., 2014; Tiklova et al., 2019; Poulin et al., 2018). Such molecularly defined subpopulations have been the subject of several recent studies. Investigations of these subtypes have ultimately unveiled many distinctive properties across several domains, such as their axonal projections and functional properties (Poulin et al., 2018; Wu et al., 2019; Pereira Luppi et al., 2021; Evans et al., 2017; Evans et al., 2020). These key differences between subtypes have begun to corroborate the biological relevance of DA neuron taxonomic schemes. We hypothesize that these putative molecular subtypes, with their distinctive circuits, could shed light on the wide variety of dopamine-related symptoms observed across several diseases including depression, chronic pain, addiction, and Parkinson's Disease. While it is difficult to reconcile how a single neurotransmitter can be involved in so many seemingly unrelated phenotypes, one solution could be the existence of several individual dopaminergic pathways serving different functions, with molecular subtypes serving as distinct nodes for these pathways. Indeed, this conceptual framework is already the dogma for anatomically distinct DA pathways, including the mesocortical, mesolimbic and mesostriatal pathways (Bjorklund & Dunnett, 2007). Here, we discuss our existing knowledge of DA neuron subtypes and attempt to provide a roadmap for how their distinctive properties can provide novel insights into the motor symptoms of Parkinson's disease (PD) (Fig. 1A). By exploring the differences between molecular subtypes and correlating this to their relative degeneration within the SNc, we may gain a deeper understanding of the cell-intrinsic mechanisms underlying why some DA neurons degenerate more than others in PD. Similarly, by mapping the inputs, projections, and functions of individual subtypes, we may better understand their individual roles in the circuit-level dysfunction of dopaminergic diseases.

Sox6 expression distinguishes dorsally and ventrally biased dopamine neurons in the substantia nigra with distinctive properties and embryonic origins.

Dopamine (DA) neurons in the ventral tier of the substantia nigra pars compacta (SNc) degenerate prominently in Parkinson's disease, while those in the dorsal tier are relatively spared. Defining the molecular, functional, and developmental characteristics of each SNc tier is crucial to understand their distinct susceptibility. We demonstrate that Sox6 expression distinguishes ventrally and dorsally biased DA neuron populations in the SNc. The Sox6+ population in the ventral SNc includes an Aldh1a1+ subset and is enriched in gene pathways that underpin vulnerability. Sox6+ neurons project to the dorsal striatum and show activity correlated with acceleration. Sox6- neurons project to the medial, ventral, and caudal striatum and respond to rewards. Moreover, we show that this adult division is encoded early in development. Overall, our work demonstrates a dual origin of the SNc that results in DA neuron cohorts with distinct molecular profiles, projections, and functions.

Information Theoretic Approaches to Deciphering the Neural Code with Functional Fluorescence Imaging.

Information theoretic metrics have proven useful in quantifying the relationship between behaviorally relevant parameters and neuronal activity with relatively few assumptions. However, these metrics are typically applied to action potential (AP) recordings and were not designed for the slow timescales and variable amplitudes typical of functional fluorescence recordings (e.g., calcium imaging). The lack of research guidelines on how to apply and interpret these metrics with fluorescence traces means the neuroscience community has yet to realize the power of information theoretic metrics. Here, we used computational methods to create mock AP traces with known amounts of information. From these, we generated fluorescence traces and examined the ability of different information metrics to recover the known information values. We provide guidelines for how to use information metrics when applying them to functional fluorescence and demonstrate their appropriate application to GCaMP6f population recordings from mouse hippocampal neurons imaged during virtual navigation.

Behavior determines the hippocampal spatial mapping of a multisensory environment.

Animals behave in multisensory environments guided by various modalities of spatial information. Mammalian navigation engages a cognitive map of space in the hippocampus. Yet it is unknown whether and how this map incorporates multiple modalities of spatial information. Here, we establish two behavioral tasks in which mice navigate the same multisensory virtual environment by either pursuing a visual landmark or tracking an odor gradient. These tasks engage different proportions of visuo-spatial and olfacto-spatial mapping CA1 neurons and different population-level representations of each sensory-spatial coordinate. Switching between tasks results in global remapping. In a third task, mice pursue a target of varying sensory modality, and this engages modality-invariant neurons mapping the abstract behaviorally relevant coordinate irrespective of its physical modality. These findings demonstrate that the hippocampus does not necessarily map space as one coherent physical variable but as a combination of sensory and abstract reference frames determined by the subject's behavioral goal.

The functional organization of excitatory synaptic input to place cells.

Hippocampal place cells contribute to mammalian spatial navigation and memory formation. Numerous models have been proposed to explain the location-specific firing of this cognitive representation, but the pattern of excitatory synaptic input leading to place firing is unknown, leaving no synaptic-scale explanation of place coding. Here we used resonant scanning two-photon microscopy to establish the pattern of synaptic glutamate input received by CA1 place cells in behaving mice. During traversals of the somatic place field, we found increased excitatory dendritic input, mainly arising from inputs with spatial tuning overlapping the somatic field, and functional clustering of this input along the dendrites over ~10 µm. These results implicate increases in total excitatory input and co-activation of anatomically clustered synaptic input in place firing. Since they largely inherit their fields from upstream synaptic partners with similar fields, many CA1 place cells appear to be part of multi-brain-region cell assemblies forming representations of specific locations.

Inactivation of the Medial Entorhinal Cortex Selectively Disrupts Learning of Interval Timing.

The entorhinal-hippocampal circuit can encode features of elapsed time, but nearly all previous research focused on neural encoding of "implicit time." Recent research has revealed encoding of "explicit time" in the medial entorhinal cortex (MEC) as mice are actively engaged in an interval timing task. However, it is unclear whether the MEC is required for temporal perception and/or learning during such explicit timing tasks. We therefore optogenetically inactivated the MEC as mice learned an interval timing "door stop" task that engaged mice in immobile interval timing behavior and locomotion-dependent navigation behavior. We find that the MEC is critically involved in learning of interval timing but not necessary for estimating temporal duration after learning. Together with our previous research, these results suggest that activity of a subcircuit in the MEC that encodes elapsed time during immobility is necessary for learning interval timing behaviors.

Multicolor Polymeric Nanoparticle Neuronal Tracers.

Deciphering the targets of axonal projections plays a pivotal role in interpreting neuronal function and pathology. Neuronal tracers are indispensable tools for uncovering the functions and interactions between different subregions of the brain. However, the selection of commercially available neuronal tracers is limited, currently comprising small molecule dyes, viruses, and a handful of synthetic nanoparticles. Here, we describe a series of polymer-based nanoparticles capable of retrograde transport along neurons in vivo in mice. These polymeric nanoparticle neuronal tracers (NNTs) are prepared with a palette of fluorescent labels. The morphologies, charges, and optical properties of NNTs are characterized by analytical methods including fluorescence microscopy, electron microscopy, and dynamic light scattering. Cytotoxicity and cellular uptake were investigated to analyze cellular interactions in vitro. Regardless of the type of fluorophore used in labeling, each tracer was of similar morphology, size, and charge and was competent for retrograde transport in vivo. The platform provides a convenient, scalable synthetic approach for nonviral tracers labeled with a range of fluorophores for in vivo neuronal projection mapping.

Navigating Through Time: A Spatial Navigation Perspective on How the Brain May Encode Time.

Interval timing, which operates on timescales of seconds to minutes, is distributed across multiple brain regions and may use distinct circuit mechanisms as compared to millisecond timing and circadian rhythms. However, its study has proven difficult, as timing on this scale is deeply entangled with other behaviors. Several circuit and cellular mechanisms could generate sequential or ramping activity patterns that carry timing information. Here we propose that a productive approach is to draw parallels between interval timing and spatial navigation, where direct analogies can be made between the variables of interest and the mathematical operations necessitated. Along with designing experiments that isolate or disambiguate timing behavior from other variables, new techniques will facilitate studies that directly address the neural mechanisms that are responsible for interval timing.

Coordination of rapid cholinergic and dopaminergic signaling in striatum during spontaneous movement.

Interplay between dopaminergic and cholinergic neuromodulation in the striatum is crucial for movement control, with prominent models proposing pro-kinetic and anti-kinetic effects of dopamine and acetylcholine release, respectively. However, the natural, movement-related signals of striatum cholinergic neurons and their relationship to simultaneous variations in dopamine signaling are unknown. Here, functional optical recordings in mice were used to establish rapid cholinergic signals in dorsal striatum during spontaneous movements. Bursts across the cholinergic population occurred at transitions between movement states and were marked by widespread network synchronization which diminished during sustained locomotion. Simultaneous cholinergic and dopaminergic recordings revealed distinct but coordinated sub-second signals, suggesting a new model where cholinergic population synchrony signals rapid changes in movement states while dopamine signals the drive to enact or sustain those states.

Dendritic mechanisms of hippocampal place field formation.

Place cells in the hippocampus are thought to form a cognitive map of space and a memory of places. How this map forms when animals are exposed to novel environments has been the subject of a great deal of research. Numerous technical advances over the past decade greatly increased our understanding of the precise mechanisms underlying place field formation. In particular, it is now possible to connect cellular and circuit mechanisms of integration, firing, and plasticity discovered in brain slices, to processes taking place in vivo as animals learn and encode novel environments. Here, we focus on recent results and describe the dendritic mechanisms most likely responsible for the formation of place fields. We also discuss key open questions that are likely to be answered in the coming years.

Evidence for a subcircuit in medial entorhinal cortex representing elapsed time during immobility.

The medial entorhinal cortex (MEC) is known to contain spatial encoding neurons that likely contribute to encoding spatial aspects of episodic memories. However, little is known about the role MEC plays in encoding temporal aspects of episodic memories, particularly during immobility. Here using a virtual 'Door Stop' task for mice, we show that MEC contains a representation of elapsed time during immobility, with individual time-encoding neurons activated at a specific moment during the immobile interval. This representation consisted of a sequential activation of time-encoding neurons and displayed variations in progression speed that correlated with variations in mouse timing behavior. Time- and space-encoding neurons were preferentially active during immobile and locomotion periods, respectively, were anatomically clustered with respect to each other, and preferentially encoded the same variable across tasks or environments. These results suggest the existence of largely non-overlapping subcircuits in MEC encoding time during immobility or space during locomotion.

Mapping projections of molecularly defined dopamine neuron subtypes using intersectional genetic approaches.

Midbrain dopamine (DA) neurons have diverse functions that can in part be explained by their heterogeneity. Although molecularly distinct subtypes of DA neurons have been identified by single-cell gene expression profiling, fundamental features such as their projection patterns have not been elucidated. Progress in this regard has been hindered by the lack of genetic tools for studying DA neuron subtypes. Here we develop intersectional genetic labeling strategies, based on combinatorial gene expression, to map the projections of molecularly defined DA neuron subtypes. We reveal distinct genetically defined dopaminergic pathways arising from the substantia nigra pars compacta and from the ventral tegmental area that innervate specific regions of the caudate putamen, nucleus accumbens and amygdala. Together, the genetic toolbox and DA neuron subtype projections presented here constitute a resource that will accelerate the investigation of this clinically significant neurotransmitter system.

An olfactory virtual reality system for mice.

All motile organisms use spatially distributed chemical features of their surroundings to guide their behaviors, but the neural mechanisms underlying such behaviors in mammals have been difficult to study, largely due to the technical challenges of controlling chemical concentrations in space and time during behavioral experiments. To overcome these challenges, we introduce a system to control and maintain an olfactory virtual landscape. This system uses rapid flow controllers and an online predictive algorithm to deliver precise odorant distributions to head-fixed mice as they explore a virtual environment. We establish an odor-guided virtual navigation behavior that engages hippocampal CA1 "place cells" that exhibit similar properties to those previously reported for real and visual virtual environments, demonstrating that navigation based on different sensory modalities recruits a similar cognitive map. This method opens new possibilities for studying the neural mechanisms of olfactory-driven behaviors, multisensory integration, innate valence, and low-dimensional sensory-spatial processing.

Increased Prevalence of Calcium Transients across the Dendritic Arbor during Place Field Formation.

Hippocampal place cell ensembles form a cognitive map of space during exposure to novel environments. However, surprisingly little evidence exists to support the idea that synaptic plasticity in place cells is involved in forming new place fields. Here we used high-resolution functional imaging to determine the signaling patterns in CA1 soma, dendrites, and axons associated with place field formation when mice are exposed to novel virtual environments. We found that putative local dendritic spikes often occur prior to somatic place field firing. Subsequently, the first occurrence of somatic place field firing was associated with widespread regenerative dendritic events, which decreased in prevalence with increased novel environment experience. This transient increase in regenerative events was likely facilitated by a reduction in dendritic inhibition. Since regenerative dendritic events can provide the depolarization necessary for Hebbian potentiation, these results suggest that activity-dependent synaptic plasticity underlies the formation of many CA1 place fields.