D1 and D2 medium spiny neurons in the nucleus accumbens core have distinct and valence-independent roles in learning.

At the core of value-based learning is the nucleus accumbens (NAc). D1- and D2-receptor-containing medium spiny neurons (MSNs) in the NAc core are hypothesized to have opposing valence-based roles in behavior. Using optical imaging and manipulation approaches in mice, we show that neither D1 nor D2 MSNs signal valence. D1 MSN responses were evoked by stimuli regardless of valence or contingency. D2 MSNs were evoked by both cues and outcomes, were dynamically changed with learning, and tracked valence-free prediction error at the population and individual neuron level. Finally, D2 MSN responses to cues were necessary for associative learning. Thus, D1 and D2 MSNs work in tandem, rather than in opposition, by signaling specific properties of stimuli to control learning.

Food Restriction Level and Reinforcement Schedule Differentially Influence Behavior during Acquisition and Devaluation Procedures in Mice.

Behavioral strategies are often classified based on whether reinforcer value controls reinforcement. Value-sensitive behaviors, in which animals update their actions when reinforcer value is changed, are classified as goal-directed; conversely, value-insensitive actions, where behavior remains consistent when the reinforcer is removed or devalued, are considered habitual. Basic reinforcement schedules can help to bias behavior toward either process: random ratio (RR) schedules are thought to promote the formation of goal-directed behaviors while random intervals (RIs) promote habitual control. However, how the schedule-specific features of these tasks interact with other factors that influence learning to control behavior has not been well characterized. Using male and female mice, we asked how distinct food restriction levels, a strategy often used to increase task engagement, interact with RR and RI schedules to control performance during task acquisition and devaluation procedures. We determined that food restriction level has a stronger effect on the behavior of mice following RR schedules compared with RI schedules, and that it promotes a decrease in response rate during devaluation procedures that is best explained by the effects of extinction rather than devaluation. Surprisingly, food restriction accelerated the decrease in response rates observed following devaluation across sequential extinction sessions, but not within a single session. Our results support the idea that the relationships between schedules and behavioral control strategies are not clear-cut and suggest that an animal's engagement in a task must be accounted for, together with the structure of reinforcement schedules, to appropriately interpret the cognitive underpinnings of behavior.

Sensitivity to outcome devaluation in operant tasks is better predicted by food restriction level than reinforcement training schedule in mice.

Behavioral strategies are often classified based on whether reinforcement is controlled by the value of the reinforcer. Value-sensitive behaviors, in which animals update their actions when reinforcer value is changed, are classified as goal-directed; conversely, value-insensitive actions, where behavior remains consistent when the reinforcer is removed or devalued, are considered habitual. Understanding the features of operant training that bias behavioral control toward either strategy is essential to understanding the cognitive and neuronal processes on which they rely. Using basic reinforcement principles, behavior can be biased toward relying on either process: random ratio (RR) schedules are thought to promote the formation of goal-directed behaviors while random intervals (RI) promote habitual control. However, how the schedule-specific features of these task structures relate to external factors to influence behavior is not well understood. Using male and female mice on distinct food restriction levels, we trained each group on RR schedules with responses-per-reinforcer rates matched to their RI counterparts to control for differences in reinforcement rate. We determined that food restriction level has a stronger effect on the behavior of mice following RR schedules than mice following RI schedules and that food restriction better predicted sensitivity to outcome devaluation than training schedule. Our results support the idea the relationships between RR or RI schedules with goal-directed or habitual behaviors, respectively, are more nuanced than previously appreciated and suggest that an animal's engagement in a task must be accounted for, together with the structure of reinforcement schedules, to appropriately interpret the cognitive underpinnings of behavior.

The transcriptional response to acute cocaine is inverted in male mice with a history of cocaine self-administration and withdrawal throughout the mesocorticolimbic system.

A large body of work has demonstrated that cocaine-induced changes in transcriptional regulation play a central role in the onset and maintenance of cocaine use disorder. An underappreciated aspect of this area of research, however, is that the pharmacodynamic properties of cocaine can change depending on an organism's previous drug-exposure history. In this study, we utilized RNA sequencing to characterize how the transcriptome-wide effects of acute cocaine exposure were altered by a history of cocaine self-administration and long-term withdrawal (30 days) in the ventral tegmental area (VTA), nucleus accumbens (NAc), and prefrontal cortex (PFC) in male mice. First, we found that the gene expression patterns induced by a single cocaine injection (10 mg/kg) were discordant between cocaine-naïve mice and mice in withdrawal from cocaine self-administration. Specifically, the same genes that were upregulated by acute cocaine in cocaine-naïve mice were downregulated by the same dose of cocaine in mice undergoing long-term withdrawal; the same pattern of opposite regulation was observed for the genes downregulated by initial acute cocaine exposure. When we analyzed this dataset further, we found that the gene expression patterns that were induced by long-term withdrawal from cocaine self-administration showed a high degree of overlap with the gene expression patterns of acute cocaine exposure - even though animals had not consumed cocaine in 30 days. Interestingly, cocaine re-exposure at this withdrawal time point reversed this expression pattern. Finally, we found that this pattern was similar across the VTA, PFC, NAc, and within each brain region the same genes were induced by acute cocaine, re-induced during long-term withdrawal, and reversed by cocaine re-exposure. Together, we identified a longitudinal pattern of gene regulation that is conserved across the VTA, PFC, and NAc, and characterized the genes constituting this pattern in each brain region.

Task parameters influence operant response variability in mice.

RATIONALE: During operant conditioning, animals associate actions with outcomes. However, patterns and rates of operant responding change over learning, which makes it difficult to distinguish changes in learning from general changes in performance or movement. Thus, understanding how task parameters influence movement execution is essential. OBJECTIVES: To understand how specific operant task parameters influenced the repetition of future operant responses, we investigated the ability of operant conditioning schedules and contingencies to promote reproducible bouts of five lever presses in mice. METHODS: Mice were trained on one of the four operant tasks to test three distinct hypotheses: (1) whether a cue presented concurrently with sucrose delivery influenced the pattern of lever pressing; (2) whether requiring animals to collect earned sucrose promoted the organization of responses into bouts; and (3) whether only reinforcing bouts where interresponse time (IRT) variances were below a target promoted reproducible patterns of operant behavior. RESULTS: (1) Signaling reinforcer delivery with a cue increased learning rates but resulted in mice pressing the lever in fast succession until the cue turned on, rather than executing discrete bouts. (2) Requiring mice to collect the reinforcer between bouts had little effect on behavior. (3) A training strategy that directly reinforced bouts with low variance IRTs was not more effective than a traditional fixed ratio schedule at promoting reproducible action execution. CONCLUSIONS: Together, our findings provide insights into the parameters of behavioral training that promote reproducible actions and that should be carefully selected when designing operant conditioning experiments.

Neural activity in the mouse claustrum in a cross-modal sensory selection task.

The claustrum, a subcortical nucleus forming extensive connections with the neocortex, has been implicated in sensory selection. Sensory-evoked claustrum activity is thought to modulate the neocortex's context-dependent response to sensory input. Recording from claustrum neurons while mice performed a tactile-visual sensory-selection task, we found that neurons in the anterior claustrum, including putative optotagged claustrocortical neurons projecting to the primary somatosensory cortex (S1), were rarely modulated by sensory input. Rather, they exhibited different types of direction-tuned motor responses. Furthermore, we found that claustrum neurons encoded upcoming movement during intertrial intervals and that pairs of claustrum neurons exhibiting synchronous firing were enriched for pairs preferring contralateral lick directions, suggesting that the activity of specific ensembles of similarly tuned claustrum neurons may modulate cortical activity. Chemogenetic inhibition of claustrocortical neurons decreased lick responses to inappropriate sensory stimuli. Altogether, our data indicate that the claustrum is integrated into higher-order premotor circuits recently implicated in decision-making.

Dopamine release in the nucleus accumbens core signals perceived saliency.

A large body of work has aimed to define the precise information encoded by dopaminergic projections innervating the nucleus accumbens (NAc). Prevailing models are based on reward prediction error (RPE) theory, in which dopamine updates associations between rewards and predictive cues by encoding perceived errors between predictions and outcomes. However, RPE cannot describe multiple phenomena to which dopamine is inextricably linked, such as behavior driven by aversive and neutral stimuli. We combined a series of behavioral tasks with direct, subsecond dopamine monitoring in the NAc of mice, machine learning, computational modeling, and optogenetic manipulations to describe behavior and related dopamine release patterns across multiple contingencies reinforced by differentially valenced outcomes. We show that dopamine release only conforms to RPE predictions in a subset of learning scenarios but fits valence-independent perceived saliency encoding across conditions. Here, we provide an extended, comprehensive framework for accumbal dopamine release in behavioral control.

The synaptic inputs and thalamic projections of two classes of layer 6 corticothalamic neurons in primary somatosensory cortex of the mouse.

Although corticothalamic neurons (CThNs) represent the largest source of synaptic input to thalamic neurons, their role in regulating thalamocortical interactions remains incompletely understood. CThNs in sensory cortex have historically been divided into two types, those with cell bodies in Layer 6 (L6) that project back to primary sensory thalamic nuclei and those with cell bodies in Layer 5 (L5) that project to higher-order thalamic nuclei and subcortical structures. Recently, diversity among L6 CThNs has increasingly been appreciated. In the rodent somatosensory cortex, two major classes of L6 CThNs have been identified: one projecting to the ventral posterior medial nucleus (VPM-only L6 CThNs) and one projecting to both VPM and the posterior medial nucleus (VPM/POm L6 CThNs). Using rabies-based tracing methods in mice, we asked whether these L6 CThN populations integrate similar synaptic inputs. We found that both types of L6 CThNs received local input from somatosensory cortex and thalamic input from VPM and POm. However, VPM/POm L6 CThNs received significantly more input from a number of additional cortical areas, higher order thalamic nuclei, and subcortical structures. We also found that the two types of L6 CThNs target different functional regions within the thalamic reticular nucleus (TRN). Together, our results indicate that these two types of L6 CThNs represent distinct information streams in the somatosensory cortex and suggest that VPM-only L6 CThNs regulate, via their more restricted circuits, sensory responses related to a cortical column while VPM/POm L6 CThNs, which are integrated into more widespread POm-related circuits, relay contextual information.

The Synaptic Organization of Layer 6 Circuits Reveals Inhibition as a Major Output of a Neocortical Sublamina.

The canonical cortical microcircuit has principally been defined by interlaminar excitatory connections among the six layers of the neocortex. However, excitatory neurons in layer 6 (L6), a layer whose functional organization is poorly understood, form relatively rare synaptic connections with other cortical excitatory neurons. Here, we show that the vast majority of parvalbumin inhibitory neurons in a sublamina within L6 send axons through the cortical layers toward the pia. These interlaminar inhibitory neurons receive local synaptic inputs from both major types of L6 excitatory neurons and receive stronger input from thalamocortical afferents than do neighboring pyramidal neurons. The distribution of these interlaminar interneurons and their synaptic connectivity further support a functional subdivision within the standard six layers of the cortex. Positioned to integrate local and long-distance inputs in this sublayer, these interneurons generate an inhibitory interlaminar output. These findings call for a revision to the canonical cortical microcircuit.

The development of local circuits in the neocortex: recent lessons from the mouse visual cortex.

Precise synaptic connections among neurons in the neocortex generate the circuits that underlie a broad repertoire of cortical functions including perception, learning and memory, and complex problem solving. The specific patterns and properties of these synaptic connections are fundamental to the computations cortical neurons perform. How such specificity arises in cortical circuits has remained elusive. Here, we first consider the cell-type, subcellular and synaptic specificity required for generating mature patterns of cortical connectivity and responses. Next, we focus on recent progress in understanding how the synaptic connections among excitatory cortical projection neurons are established during development using the primary visual cortex of the mouse as a model.

A Non-canonical Feedback Circuit for Rapid Interactions between Somatosensory Cortices.

Sensory perception depends on interactions among cortical areas. These interactions are mediated by canonical patterns of connectivity in which higher areas send feedback projections to lower areas via neurons in superficial and deep layers. Here, we probed the circuit basis of interactions among two areas critical for touch perception in mice, whisker primary (wS1) and secondary (wS2) somatosensory cortices. Neurons in layer 4 of wS2 (S2L4) formed a major feedback pathway to wS1. Feedback from wS2 to wS1 was organized somatotopically. Spikes evoked by whisker deflections occurred nearly as rapidly in wS2 as in wS1, including among putative S2L4 → S1 feedback neurons. Axons from S2L4 → S1 neurons sent stimulus orientation-specific activity to wS1. Optogenetic excitation of S2L4 neurons modulated activity across both wS2 and wS1, while inhibition of S2L4 reduced orientation tuning among wS1 neurons. Thus, a non-canonical feedback circuit, originating in layer 4 of S2, rapidly modulates early tactile processing.

Variation in Activity State, Axonal Projection, and Position Define the Transcriptional Identity of Individual Neocortical Projection Neurons.

Single-cell RNA sequencing has generated catalogs of transcriptionally defined neuronal subtypes of the brain. However, the cellular processes that contribute to neuronal subtype specification and transcriptional heterogeneity remain unclear. By comparing the gene expression profiles of single layer 6 corticothalamic neurons in somatosensory cortex, we show that transcriptional subtypes primarily reflect axonal projection pattern, laminar position within the cortex, and neuronal activity state. Pseudotemporal ordering of 1,023 cellular responses to sensory manipulation demonstrates that changes in expression of activity-induced genes both reinforced cell-type identity and contributed to increased transcriptional heterogeneity within each cell type. This is due to cell-type biased choices of transcriptional states following manipulation of neuronal activity. These results reveal that axonal projection pattern, laminar position, and activity state define significant axes of variation that contribute both to the transcriptional identity of individual neurons and to the transcriptional heterogeneity within each neuronal subtype.

Multicluster Pcdh diversity is required for mouse olfactory neural circuit assembly.

The vertebrate clustered protocadherin (Pcdh) cell surface proteins are encoded by three closely linked gene clusters (Pcdhα, Pcdhß, and Pcdhγ). Here, we show that all three gene clusters functionally cooperate to provide individual mouse olfactory sensory neurons (OSNs) with the cell surface diversity required for their assembly into distinct glomeruli in the olfactory bulb. Although deletion of individual Pcdh clusters had subtle phenotypic consequences, the loss of all three clusters (tricluster deletion) led to a severe axonal arborization defect and loss of self-avoidance. By contrast, when endogenous Pcdh diversity is overridden by the expression of a single-tricluster gene repertoire (α and ß and γ), OSN axons fail to converge to form glomeruli, likely owing to contact-mediated repulsion between axons expressing identical combinations of Pcdh isoforms.

Molecular logic of neuronal self-recognition through protocadherin domain interactions.

Self-avoidance, a process preventing interactions of axons and dendrites from the same neuron during development, is mediated in vertebrates through the stochastic single-neuron expression of clustered protocadherin protein isoforms. Extracellular cadherin (EC) domains mediate isoform-specific homophilic binding between cells, conferring cell recognition through a poorly understood mechanism. Here, we report crystal structures for the EC1-EC3 domain regions from four protocadherin isoforms representing the α, ß, and γ subfamilies. All are rod shaped and monomeric in solution. Biophysical measurements, cell aggregation assays, and computational docking reveal that trans binding between cells depends on the EC1-EC4 domains, which interact in an antiparallel orientation. We also show that the EC6 domains are required for the formation of cis-dimers. Overall, our results are consistent with a model in which protocadherin cis-dimers engage in a head-to-tail interaction between EC1-EC4 domains from apposed cell surfaces, possibly forming a zipper-like protein assembly, and thus providing a size-dependent self-recognition mechanism.

Single-cell identity generated by combinatorial homophilic interactions between α, ß, and γ protocadherins.

Individual mammalian neurons stochastically express distinct repertoires of α, ß, and γ protocadherin (Pcdh) proteins, which function in neural circuit assembly. We report that all three subfamilies of clustered Pcdhs can engage in specific homophilic interactions, that cell surface delivery of Pcdhα isoforms requires cis interactions with other Pcdhs, and that the extracellular cadherin domain EC6 plays a critical role in this process. Examination of homophilic interactions between specific combinations of multiple Pcdh isoforms revealed that Pcdh combinatorial recognition specificities depend on the identity of all of the expressed isoforms. A single mismatched Pcdh isoform can interfere with these combinatorial homophilic interactions. A theoretical analysis reveals that assembly of Pcdh isoforms into multimeric recognition units and the observed tolerance for mismatched isoforms can generate cell surface diversity sufficient for single-cell identity. However, the competing demands of nonself discrimination and self-recognition place limitations on the mechanisms by which homophilic recognition units can function.