Whole-brain spatial organization of hippocampal single-neuron projectomes.

Mapping single-neuron projections is essential for understanding brain-wide connectivity and diverse functions of the hippocampus (HIP). Here, we reconstructed 10,100 single-neuron projectomes of mouse HIP and classified 43 projectome subtypes with distinct projection patterns. The number of projection targets and axon-tip distribution depended on the soma location along HIP longitudinal and transverse axes. Many projectome subtypes were enriched in specific HIP subdomains defined by spatial transcriptomic profiles. Furthermore, we delineated comprehensive wiring diagrams for HIP neurons projecting exclusively within the HIP formation (HPF) and for those projecting to both intra- and extra-HPF targets. Bihemispheric projecting neurons generally projected to one pair of homologous targets with ipsilateral preference. These organization principles of single-neuron projectomes provide a structural basis for understanding the function of HIP neurons.

High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.

Deciphering patterns of connectivity between neurons in the brain is a critical step toward understanding brain function. Imaging-based neuroanatomical tracing identifies area-to-area or sparse neuron-to-neuron connectivity patterns, but with limited throughput. Barcode-based connectomics maps large numbers of single-neuron projections, but remains a challenge for jointly analyzing single-cell transcriptomics. Here, we established a rAAV2-retro barcode-based multiplexed tracing method that simultaneously characterizes the projectome and transcriptome at the single neuron level. We uncovered dedicated and collateral projection patterns of ventromedial prefrontal cortex (vmPFC) neurons to five downstream targets and found that projection-defined vmPFC neurons are molecularly heterogeneous. We identified transcriptional signatures of projection-specific vmPFC neurons, and verified Pou3f1 as a marker gene enriched in neurons projecting to the lateral hypothalamus, denoting a distinct subset with collateral projections to both dorsomedial striatum and lateral hypothalamus. In summary, we have developed a new multiplexed technique whose paired connectome and gene expression data can help reveal organizational principles that form neural circuits and process information.

Prefrontal projections modulate recurrent circuitry in the insular cortex to support short-term memory.

Short-term memory (STM) maintains information during a short delay period. How long-range and local connections interact to support STM encoding remains elusive. Here, we tackle the problem focusing on long-range projections from the medial prefrontal cortex (mPFC) to the anterior agranular insular cortex (aAIC) in head-fixed mice performing an olfactory delayed-response task. Optogenetic and electrophysiological experiments reveal the behavioral importance of the two regions in encoding STM information. Spike-correlogram analysis reveals strong local and cross-region functional coupling (FC) between memory neurons encoding the same information. Optogenetic suppression of mPFC-aAIC projections during the delay period reduces behavioral performance, the proportion of memory neurons, and memory-specific FC within the aAIC, whereas optogenetic excitation enhances all of them. mPFC-aAIC projections also bidirectionally modulate the efficacy of STM-information transfer, measured by the contribution of FC spiking pairs to the memory-coding ability of following neurons. Thus, prefrontal projections modulate insular neurons' functional connectivity and memory-coding ability to support STM.

A brain-inspired algorithm that mitigates catastrophic forgetting of artificial and spiking neural networks with low computational cost.

Neuromodulators in the brain act globally at many forms of synaptic plasticity, represented as metaplasticity, which is rarely considered by existing spiking (SNNs) and nonspiking artificial neural networks (ANNs). Here, we report an efficient brain-inspired computing algorithm for SNNs and ANNs, referred to here as neuromodulation-assisted credit assignment (NACA), which uses expectation signals to induce defined levels of neuromodulators to selective synapses, whereby the long-term synaptic potentiation and depression are modified in a nonlinear manner depending on the neuromodulator level. The NACA algorithm achieved high recognition accuracy with substantially reduced computational cost in learning spatial and temporal classification tasks. Notably, NACA was also verified as efficient for learning five different class continuous learning tasks with varying degrees of complexity, exhibiting a markedly mitigated catastrophic forgetting at low computational cost. Mapping synaptic weight changes showed that these benefits could be explained by the sparse and targeted synaptic modifications attributed to expectation-based global neuromodulation.

Single-neuron projectome of mouse prefrontal cortex.

Prefrontal cortex (PFC) is the cognitive center that integrates and regulates global brain activity. However, the whole-brain organization of PFC axon projections remains poorly understood. Using single-neuron reconstruction of 6,357 mouse PFC projection neurons, we identified 64 projectome-defined subtypes. Each of four previously known major cortico-cortical subnetworks was targeted by a distinct group of PFC subtypes defined by their first-order axon collaterals. Further analysis unraveled topographic rules of soma distribution within PFC, first-order collateral branch point-dependent target selection and terminal arbor distribution-dependent target subdivision. Furthermore, we obtained a high-precision hierarchical map within PFC and three distinct functionally related PFC modules, each enriched with internal recurrent connectivity. Finally, we showed that each transcriptome subtype corresponds to multiple projectome subtypes found in different PFC subregions. Thus, whole-brain single-neuron projectome analysis reveals organization principles of axon projections within and outside PFC and provides the essential basis for elucidating neuronal connectivity underlying diverse PFC functions.

Hierarchy in sensory processing reflected by innervation balance on cortical interneurons.

Sensory processing is subjected to modulation by behavioral contexts that are often mediated by long-range inputs to cortical interneurons, but their selectivity to different types of interneurons remains largely unknown. Using rabies-virus tracing and optogenetics-assisted recording, we analyzed the long-range connections to various brain regions along the hierarchy of visual processing, including primary visual cortex, medial association cortices, and frontal cortices. We found that hierarchical corticocortical and thalamocortical connectivity is reflected by the relative weights of inputs to parvalbumin-positive (PV+) and vasoactive intestinal peptide-positive (VIP+) neurons within the conserved local circuit motif, with bottom-up and top-down inputs preferring PV+ and VIP+ neurons, respectively. Our algorithms based on innervation weights for these two types of local interneurons generated testable predictions of the hierarchical position of many brain areas. These results support the notion that preferential long-range inputs to specific local interneurons are essential for the hierarchical information flow in the brain.

Transient Delay-Period Activity of Agranular Insular Cortex Controls Working Memory Maintenance in Learning Novel Tasks.

Whether transient or sustained neuronal activity during the delay period underlies working memory (WM) has been debated. Here, we report that transient, but not sustained, delay-period activity in mouse anterior agranular insular cortex (aAIC) plays a dominant role in maintaining WM information during learning of novel olfactory tasks. By optogenetic screening over 12 brain regions, we found that suppressing aAIC activity markedly impaired olfactory WM maintenance during learning. Single-unit recording showed that odor-selective aAIC neurons with predominantly transient firing patterns encoded WM information. Both WM task performance and transient-neuron proportion were enhanced and reduced by activating and suppressing the delay-period activity of the projection from medial prefrontal cortex (mPFC) to aAIC. The ability of mice to resist delay-period distractors also correlated with an increased percentage of transient neurons. Therefore, transient, but not sustained, aAIC neuronal activity during the delay period is largely responsible for maintaining information while learning novel WM tasks.

Active information maintenance in working memory by a sensory cortex.

Working memory is a critical brain function for maintaining and manipulating information over delay periods of seconds. It is debated whether delay-period neural activity in sensory regions is important for the active maintenance of information during the delay period. Here, we tackle this question by examining the anterior piriform cortex (APC), an olfactory sensory cortex, in head-fixed mice performing several olfactory working memory tasks. Active information maintenance is necessary in these tasks, especially in a dual-task paradigm in which mice are required to perform another distracting task while actively maintaining information during the delay period. Optogenetic suppression of neuronal activity in APC during the delay period impaired performance in all the tasks. Furthermore, electrophysiological recordings revealed that APC neuronal populations encoded odor information in the delay period even with an intervening distracting task. Thus, delay activity in APC is important for active information maintenance in olfactory working memory.

Awake and behaving mouse fMRI during Go/No-Go task.

Functional magnetic imaging (fMRI) has been widely used to examine the functional neural networks in both the evoked and resting states. However, most fMRI studies in rodents are performed under anesthesia, which greatly limits the scope of their application, and behavioral relevance. Efforts have been made to image rodents in the awake condition, either in the resting state or in response to sensory or optogenetic stimulation. However, fMRI in awake behaving rodents has not yet been achieved. In the current study, a novel fMRI paradigm for awake and behaving mice was developed, allowing functional imaging of the mouse brain in an olfaction-based go/no-go task. High resolution functional imaging with limited motion and image distortion were achieved at 9.4T with a cryogenic coil in awake and behaving mice. Distributed whole-brain spatiotemporal patterns were revealed, with drastically different activity profiles for go versus no-go trials. Therefore, we have demonstrated the feasibility of functional imaging of an olfactory behavior in awake mice. This fMRI paradigm in awake behaving mice could lead to novel insights into neural mechanisms underlying behaviors at a whole-brain level.

High-Throughput Automatic Training System for Odor-Based Learned Behaviors in Head-Fixed Mice.

Understanding neuronal mechanisms of learned behaviors requires efficient behavioral assays. We designed a high-throughput automatic training system (HATS) for olfactory behaviors in head-fixed mice. The hardware and software were constructed to enable automatic training with minimal human intervention. The integrated system was composed of customized 3D-printing supporting components, an odor-delivery unit with fast response, Arduino based hardware-controlling and data-acquisition unit. Furthermore, the customized software was designed to enable automatic training in all training phases, including lick-teaching, shaping and learning. Using HATS, we trained mice to perform delayed non-match to sample (DNMS), delayed paired association (DPA), Go/No-go (GNG), and GNG reversal tasks. These tasks probed cognitive functions including sensory discrimination, working memory, decision making and cognitive flexibility. Mice reached stable levels of performance within several days in the tasks. HATS enabled an experimenter to train eight mice simultaneously, therefore greatly enhanced the experimental efficiency. Combined with causal perturbation and activity recording techniques, HATS can greatly facilitate our understanding of the neural-circuitry mechanisms underlying learned behaviors.

Medial prefrontal activity during delay period contributes to learning of a working memory task.

Cognitive processes require working memory (WM) that involves a brief period of memory retention known as the delay period. Elevated delay-period activity in the medial prefrontal cortex (mPFC) has been observed, but its functional role in WM tasks remains unclear. We optogenetically suppressed or enhanced activity of pyramidal neurons in mouse mPFC during the delay period. Behavioral performance was impaired during the learning phase but not after the mice were well trained. Delay-period mPFC activity appeared to be more important in memory retention than in inhibitory control, decision-making, or motor selection. Furthermore, endogenous delay-period mPFC activity showed more prominent modulation that correlated with memory retention and behavioral performance. Thus, properly regulated mPFC delay-period activity is critical for information retention during learning of a WM task.