Automating licking bias correction in a two-choice delayed match-to-sample task to accelerate learning.

Animals often display choice bias, or a preference for one option over the others, which can significantly impede learning new tasks. Delayed match-to-sample (DMS) tasks with two-alternative choices of lickports on the left and right have been widely used to study sensory processing, working memory, and associative memory in head-fixed animals. However, extensive training time, primarily due to the animals' biased licking responses, limits their practical utility. Here, we present the implementation of an automated side bias correction system in an olfactory DMS task, where the lickport positions and the ratio of left- and right-rewarded trials are dynamically adjusted to counterbalance mouse's biased licking responses during training. The correction algorithm moves the preferred lickport farther away from the mouse's mouth and the non-preferred lickport closer, while also increasing the proportion of non-preferred side trials when biased licking occurs. We found that adjusting lickport distances and the proportions of left- versus right-rewarded trials effectively reduces the mouse's side bias. Further analyses reveal that these adjustments also correlate with subsequent improvements in behavioral performance. Our findings suggest that the automated side bias correction system is a valuable tool for enhancing the applicability of behavioral tasks involving two-alternative lickport choices.

Sensory cortical ensembles exhibit differential coupling to ripples in distinct hippocampal subregions.

Cortical neurons activated during recent experiences often reactivate with dorsal hippocampal CA1 ripples during subsequent rest. Less is known about cortical interactions with intermediate hippocampal CA1, whose connectivity, functions, and ripple events differ from dorsal CA1. We identified three clusters of putative excitatory neurons in mouse visual cortex that are preferentially excited together with either dorsal or intermediate CA1 ripples or suppressed before both ripples. Neurons in each cluster were evenly distributed across primary and higher visual cortices and co-active even in the absence of ripples. These ensembles exhibited similar visual responses but different coupling to thalamus and pupil-indexed arousal. We observed a consistent activity sequence preceding and predicting ripples: (1) suppression of ripple-suppressed cortical neurons, (2) thalamic silence, and (3) activation of intermediate CA1-ripple-activated cortical neurons. We propose that coordinated dynamics of these ensembles relay visual experiences to distinct hippocampal subregions for incorporation into different cognitive maps.

Reward learning improves social signal processing in autism model mice.

Social and reward signal processing and their association are critical elements of social motivation. Despite the use of reward learning to improve the social interactions of patients with autism spectrum disorder (ASD), the underlying neural mechanisms are unknown. Here, we found different yet conjunct neuronal representations of social and reward signals in the mouse medial prefrontal cortex (mPFC). We also found that social signal processing is selectively disrupted, whereas reward signal processing is intact in the mPFC of Shank2-knockout mice, a mouse model of ASD. Furthermore, reward learning not only allows Shank2-knockout mice to associate social stimuli with reward availability, but it also rescues the impaired social signal processing. These findings provide insights into the neural basis for the therapeutic use of reward learning in ASD.

Protocol for calcium imaging of dorsal and ventral CA1 neurons in head-fixed mice.

In contrast to other techniques utilized in physiological studies, calcium imaging can visualize target neurons located deep in the brain. Here, we present a protocol for one-photon calcium imaging of dorsal and ventral CA1 neurons in head-fixed mice. We describe procedures for injecting GCaMP6f virus, implanting a gradient-index (GRIN) lens, and installing a baseplate for Inscopix microscope mounting. For complete details on the use and execution of this protocol, please refer to Yun et al.1.

Sensory cortical ensembles exhibit differential coupling to ripples in distinct hippocampal subregions.

Cortical neurons activated during recent experiences often reactivate with dorsal hippocampal CA1 sharp-wave ripples (SWRs) during subsequent rest. Less is known about cortical interactions with intermediate hippocampal CA1, whose connectivity, functions, and SWRs differ from those of dorsal CA1. We identified three clusters of visual cortical excitatory neurons that are excited together with either dorsal or intermediate CA1 SWRs, or suppressed before both SWRs. Neurons in each cluster were distributed across primary and higher visual cortices and co-active even in the absence of SWRs. These ensembles exhibited similar visual responses but different coupling to thalamus and pupil-indexed arousal. We observed a consistent activity sequence: (i) suppression of SWR-suppressed cortical neurons, (ii) thalamic silence, and (iii) activation of the cortical ensemble preceding and predicting intermediate CA1 SWRs. We propose that the coordinated dynamics of these ensembles relay visual experiences to distinct hippocampal subregions for incorporation into different cognitive maps.

Septotemporal variations in hippocampal value and outcome processing.

A large body of evidence indicates functional variations along the hippocampal longitudinal axis. To investigate whether and how value and outcome processing vary between the dorsal (DH) and the ventral hippocampus (VH), we examined neuronal activity and inactivation effects of the DH and VH in mice performing probabilistic classical conditioning tasks. Inactivation of either structure disrupts value-dependent anticipatory licking, and value-coding neurons are found in both structures, indicating their involvement in value processing. However, the DH neuronal population increases activity as a function of value, while the VH neuronal population is preferentially responsive to the highest-value sensory cue. Also, signals related to outcome-dependent value learning are stronger in the DH. VH neurons instead show rapid responses to punishment and strongly biased responses to negative prediction error. These findings suggest that the DH faithfully represents the external value landscape, whereas the VH preferentially represents behaviorally relevant, salient features of experienced events.

Suppressed prefrontal neuronal firing variability and impaired social representation in IRSp53-mutant mice.

Social deficit is a major feature of neuropsychiatric disorders, including autism spectrum disorders, schizophrenia, and attention-deficit/hyperactivity disorder, but its neural mechanisms remain unclear. Here, we examined neuronal discharge characteristics in the medial prefrontal cortex (mPFC) of IRSp53/Baiap2-mutant mice, which show social deficits, during social approach. We found a decrease in the proportion of IRSp53-mutant excitatory mPFC neurons encoding social information, but not that encoding non-social information. In addition, the firing activity of IRSp53-mutant neurons was less differential between social and non-social targets. IRSp53-mutant excitatory mPFC neurons displayed an increase in baseline neuronal firing, but decreases in the variability and dynamic range of firing as well as burst firing during social and non-social target approaches compared to wild-type controls. Treatment of memantine, an NMDA receptor antagonist that rescues social deficit in IRSp53-mutant mice, alleviates the reduced burst firing of IRSp53-mutant pyramidal mPFC neurons. These results suggest that suppressed neuronal activity dynamics and burst firing may underlie impaired cortical encoding of social information and social behaviors in IRSp53-mutant mice.

Enhanced fear limits behavioral flexibility in Shank2-deficient mice.

BACKGROUND: A core symptom of autism spectrum disorder (ASD) is repetitive and restrictive patterns of behavior. Cognitive inflexibility has been proposed as a potential basis for these symptoms of ASD. More generally, behavioral inflexibility has been proposed to underlie repetitive and restrictive behavior in ASD. Here, we investigated whether and how behavioral flexibility is compromised in a widely used animal model of ASD. METHODS: We compared the behavioral performance of Shank2-knockout mice and wild-type littermates in reversal learning employing a probabilistic classical trace conditioning paradigm. A conditioned stimulus (odor) was paired with an unconditioned appetitive (water, 6 µl) or aversive (air puff) stimulus in a probabilistic manner. We also compared air puff-induced eye closure responses of Shank2-knockout and wild-type mice. RESULTS: Male, but not female, Shank2-knockout mice showed impaired reversal learning when the expected outcomes consisted of a water reward and a strong air puff. Moreover, male, but not female, Shank2-knockout mice showed stronger anticipatory eye closure responses to the air puff compared to wild-type littermates, raising the possibility that the impairment might reflect enhanced fear. In support of this contention, male Shank2-knockout mice showed intact reversal learning when the strong air puff was replaced with a mild air puff and when the expected outcomes consisted of only rewards. LIMITATIONS: We examined behavioral flexibility in one behavioral task (reversal learning in a probabilistic classical trace conditioning paradigm) using one ASD mouse model (Shank2-knockout mice). Thus, future work is needed to clarify the extent to which our findings (that enhanced fear limits behavioral flexibility in ASD) can explain the behavioral inflexibility associated with ASD. Also, we examined only the relationship between fear and behavioral flexibility, leaving open the question of whether abnormalities in processes other than fear contribute to behavioral inflexibility in ASD. Finally, the neurobiological mechanisms linking Shank2-knockout and enhanced fear remain to be elucidated. CONCLUSIONS: Our results indicate that enhanced fear suppresses reversal learning in the presence of an intact capability to learn cue-outcome contingency changes in Shank2-knockout mice. Our findings suggest that behavioral flexibility might be seriously limited by abnormal emotional responses in ASD.

Variations in Commissural Input Processing Across Different Types of Cortical Projection Neurons.

To understand how incoming cortical inputs are processed by different types of cortical projection neurons in the medial prefrontal cortex, we compared intrinsic physiological properties of and commissural excitatory/inhibitory influences on layer 5 intratelencephalic (IT), layer 5 pyramidal tract (PT), and layers 2/3 IT projection neurons. We found that intrinsic physiological properties and commissural synaptic transmission varied across the three types of projection neurons. The rank order of intrinsic excitability was layer 5 PT > layer 5 IT > layers 2/3 IT neurons. Commissural connectivity was higher in layers 2/3 than layer 5 projection neurons, but commissural excitatory influence was stronger on layer 5 than layers 2/3 pyramidal neurons. Paired-pulse ratio was also greater in PT than IT neurons. These results indicate that commissural inputs activate deep layer PT neurons most preferentially and superficial layer IT neurons least preferentially. Deep layer PT neurons might faithfully transmit cortical input signals to downstream subcortical structures for reliable control of behavior, whereas superficial layer IT neurons might integrate cortical input signals from diverse sources in support of higher-order cognitive functions.

A role of anterior cingulate cortex in the emergence of worker-parasite relationship.

We studied the brain mechanisms underlying action selection in a social dilemma setting in which individuals' effortful gains are unfairly distributed among group members. A stable "worker-parasite" relationship developed when three individually operant-conditioned rats were placed together in a Skinner box equipped with response lever and food dispenser on opposite sides. Specifically, one rat, the "worker," engaged in lever-pressing while the other two "parasitic" rats profited from the worker's effort by crowding the feeder in anticipation of food. Anatomically, c-Fos expression in the anterior cingulate cortex (ACC) was significantly higher in worker rats than in parasite rats. Functionally, ACC inactivation suppressed the worker's lever-press behavior drastically under social, but only mildly under individual, settings. Transcriptionally, GABAA receptor- and potassium channel-related messenger RNA expressions were reliably lower in the worker's, relative to parasite's, ACC. These findings indicate the requirement of ACC activation for the expression of exploitable, effortful behavior, which could be mediated by molecular pathways involving GABAA receptor/potassium channel proteins.

Excitatory synapses and gap junctions cooperate to improve Pv neuronal burst firing and cortical social cognition in Shank2-mutant mice.

NMDA receptor (NMDAR) and GABA neuronal dysfunctions are observed in animal models of autism spectrum disorders, but how these dysfunctions impair social cognition and behavior remains unclear. We report here that NMDARs in cortical parvalbumin (Pv)-positive interneurons cooperate with gap junctions to promote high-frequency (>80 Hz) Pv neuronal burst firing and social cognition. Shank2-/- mice, displaying improved sociability upon NMDAR activation, show impaired cortical social representation and inhibitory neuronal burst firing. Cortical Shank2-/- Pv neurons show decreased NMDAR activity, which suppresses the cooperation between NMDARs and gap junctions (GJs) for normal burst firing. Shank2-/- Pv neurons show compensatory increases in GJ activity that are not sufficient for social rescue. However, optogenetic boosting of Pv neuronal bursts, requiring GJs, rescues cortical social cognition in Shank2-/- mice, similar to the NMDAR-dependent social rescue. Therefore, NMDARs and gap junctions cooperate to promote cortical Pv neuronal bursts and social cognition.

Parallel processing of working memory and temporal information by distinct types of cortical projection neurons.

It is unclear how different types of cortical projection neurons work together to support diverse cortical functions. We examined the discharge characteristics and inactivation effects of intratelencephalic (IT) and pyramidal tract (PT) neurons-two major types of cortical excitatory neurons that project to cortical and subcortical structures, respectively-in the deep layer of the medial prefrontal cortex in mice performing a delayed response task. We found stronger target-dependent firing of IT than PT neurons during the delay period. We also found the inactivation of IT neurons, but not PT neurons, impairs behavioral performance. In contrast, PT neurons carry more temporal information than IT neurons during the delay period. Our results indicate a division of labor between IT and PT projection neurons in the prefrontal cortex for the maintenance of working memory and for tracking the passage of time, respectively.

Robust and distributed neural representation of action values.

Studies in rats, monkeys, and humans have found action-value signals in multiple regions of the brain. These findings suggest that action-value signals encoded in these brain structures bias choices toward higher expected rewards. However, previous estimates of action-value signals might have been inflated by serial correlations in neural activity and also by activity related to other decision variables. Here, we applied several statistical tests based on permutation and surrogate data to analyze neural activity recorded from the striatum, frontal cortex, and hippocampus. The results show that previously identified action-value signals in these brain areas cannot be entirely accounted for by concurrent serial correlations in neural activity and action value. We also found that neural activity related to action value is intermixed with signals related to other decision variables. Our findings provide strong evidence for broadly distributed neural signals related to action value throughout the brain.

Active maintenance of eligibility trace in rodent prefrontal cortex.

Even though persistent neural activity has been proposed as a mechanism for maintaining eligibility trace, direct empirical evidence for active maintenance of eligibility trace has been lacking. We recorded neuronal activity in the medial prefrontal cortex (mPFC) in rats performing a dynamic foraging task in which a choice must be remembered until its outcome on the timescale of seconds for correct credit assignment. We found that mPFC neurons maintain significant choice signals during the time period between action selection and choice outcome. We also found that neural signals for choice, outcome, and action value converge in the mPFC when choice outcome was revealed. Our results indicate that the mPFC maintains choice signals necessary for temporal credit assignment in the form of persistent neural activity in our task. They also suggest that the mPFC might update action value by combining actively maintained eligibility trace with action value and outcome signals.

Spatial organization of functional clusters representing reward and movement information in the striatal direct and indirect pathways.

To obtain insights into striatal neural processes underlying reward-based learning and movement control, we examined spatial organizations of striatal neurons related to movement and reward-based learning. For this, we recorded the activity of direct- and indirect-pathway neurons (D1 and A2a receptor-expressing neurons, respectively) in mice engaged in probabilistic classical conditioning and open-field free exploration. We found broadly organized functional clusters of striatal neurons in the direct as well as indirect pathways for both movement- and reward-related variables. Functional clusters for different variables were partially overlapping in both pathways, but the overlap between outcome- and value-related functional clusters was greater in the indirect than direct pathway. Also, value-related spatial clusters were progressively refined during classical conditioning. Our study shows the broad and learning-dependent spatial organization of functional clusters of dorsal striatal neurons in the direct and indirect pathways. These findings further argue against the classic model of the basal ganglia and support the importance of spatiotemporal patterns of striatal neuronal ensemble activity in the control of behavior.

Somatostatin enhances visual processing and perception by suppressing excitatory inputs to parvalbumin-positive interneurons in V1.

Somatostatin (SST) is a neuropeptide expressed in a major subtype of GABAergic interneurons in the cortex. Despite abundant expression of SST and its receptors, their modulatory function in cortical processing remains unclear. Here, we found that SST application in the primary visual cortex (V1) improves visual discrimination in freely moving mice and enhances orientation selectivity of V1 neurons. We also found that SST reduced excitatory synaptic transmission to parvalbumin-positive (PV+) fast-spiking interneurons but not to regular-spiking neurons. Last, using serial block-face scanning electron microscopy (SBEM), we found that axons of SST+ neurons in V1 often contact other axons that exhibit excitatory synapses onto the soma and proximal dendrites of the PV+ neuron. Collectively, our results demonstrate that the neuropeptide SST improves visual perception by enhancing visual gain of V1 neurons via a reduction in excitatory synaptic transmission to PV+ inhibitory neurons.

Distinct roles of parvalbumin- and somatostatin-expressing neurons in flexible representation of task variables in the prefrontal cortex.

A hallmark of the prefrontal cortex (PFC) is flexible representation of task-relevant variables. To investigate roles of different interneuron subtypes in this process, we examined discharge characteristics and inactivation effects of parvalbumin (PV)- and somatostatin (SST)-expressing neurons in the mouse PFC during probabilistic classical conditioning. We found activity patterns and inactivation effects differed between PV and SST neurons: SST neurons conveyed cue-associated quantitative value signals until trial outcome, whereas PV neurons maintained valence signals even after trial outcome. Also, PV, but not SST, neuronal population showed opposite responses to reward and punishment. Moreover, inactivation of PV, but not SST, neurons affected outcome responses and activity reversal of pyramidal neurons. Modeling suggested opposite responses of PV neurons to reward and punishment as an efficient mechanism for facilitating rapid cue-outcome contingency learning. Our results suggest primary roles of mPFC PV neurons in rapid value updating and SST neurons in predicting values of upcoming events.

Transient effect of mossy fiber stimulation on spatial firing of CA3 neurons in familiar and novel environments.

Hippocampal mossy fibers have long been proposed to impose new patterns to learn onto CA3 neurons during new memory formation. However, inconsistent with this theory, we found in our previous study that mossy fiber stimulation induces only transient changes in CA3 spatial firing in a familiar environment. Here, we tested whether mossy fiber stimulation affects CA3 spatial firing differently between familiar and novel environments. We compared spatial firing of CA3 neurons before and after optogenetic stimulation of mossy fibers in freely behaving mice in a familiar and three sets of novel environments. We found that CA3 neurons are more responsive to mossy fiber stimulation in the novel than familiar environments. However, we failed to obtain evidence for long-lasting effect of mossy fiber stimulation on spatial firing of CA3 neurons in both the familiar and novel environments. Our results provide further evidence against the view that mossy fibers carry teaching signals.

Distinct effects of reward and navigation history on hippocampal forward and reverse replays.

To better understand the functional roles of hippocampal forward and reverse replays, we trained rats in a spatial sequence memory task and examined how these replays are modulated by reward and navigation history. We found that reward enhances both forward and reverse replays during the awake state, but in different ways. Reward enhances the rate of reverse replays, but it increases the fidelity of forward replays for recently traveled as well as other alternative trajectories heading toward a rewarding location. This suggests roles for forward and reverse replays in reinforcing representations for all potential rewarding trajectories. We also found more faithful reactivation of upcoming than already rewarded trajectories in forward replays. This suggests a role for forward replays in preferentially reinforcing representations for high-value trajectories. We propose that hippocampal forward and reverse replays might contribute to constructing a map of potential navigation trajectories and their associated values (a "value map") via distinct mechanisms.

Dynamically changing neuronal activity supporting working memory for predictable and unpredictable durations.

Diverse neural processes have been proposed as the neural basis of working memory. To investigate whether the medial prefrontal cortex (mPFC) relies on different neural processes to mediate working memory depending on the predictability of delay duration, we examined mPFC neural activity in mice performing a delayed response task with fixed (4 s) or random (between 1-7 s) delay durations. mPFC neural activity was strongly influenced by the predictability of delay duration. Nevertheless, mPFC neurons seldom showed persistent activity spanning the entire delay period and instead showed dynamically-changing delay-period activity under both the fixed-delay and random-delay conditions. mPFC neurons conveyed higher working memory information under the random-delay than fixed-delay conditions, possibly due to a higher demand for stable working memory maintenance. Our results suggest that the rodent mPFC may rely on dynamically-changing neuronal activity to maintain working memory regardless of the predictability of delay duration.