Multi-region calcium imaging in freely behaving mice with ultra-compact head-mounted fluorescence microscopes.

To investigate the circuit-level neural mechanisms of behavior, simultaneous imaging of neuronal activity in multiple cortical and subcortical regions is highly desired. Miniature head-mounted microscopes offer the capability of calcium imaging in freely behaving animals. However, implanting multiple microscopes on a mouse brain remains challenging due to space constraints and the cumbersome weight of the equipment. Here, we present TINIscope, a Tightly Integrated Neuronal Imaging microscope optimized for electronic and opto-mechanical design. With its compact and lightweight design of 0.43 g, TINIscope enables unprecedented simultaneous imaging of behavior-relevant activity in up to four brain regions in mice. Proof-of-concept experiments with TINIscope recorded over 1000 neurons in four hippocampal subregions and revealed concurrent activity patterns spanning across these regions. Moreover, we explored potential multi-modal experimental designs by integrating additional modules for optogenetics, electrical stimulation or local field potential recordings. Overall, TINIscope represents a timely and indispensable tool for studying the brain-wide interregional coordination that underlies unrestrained behaviors.

Preferential subcortical collateral projections of pedunculopontine nucleus-targeting cortical pyramidal neurons revealed by brain-wide single fiber tracing.

The pedunculopontine nucleus (PPN) is a heterogeneous midbrain structure involved in various brain functions, such as motor control, learning, reward, and sleep. Previous studies using conventional tracers have shown that the PPN receives extensive afferent inputs from various cortical areas. To examine how these cortical axons make collateral projections to other subcortical areas, we used a dual-viral injection strategy to sparsely label PPN-targeting cortical pyramidal neurons in CaMKIIα-Cre transgenic mice. Using a high-speed volumetric imaging with on-the-fly-scan and Readout (VISoR) technique, we visualized brain-wide axonal projections of individual PPN-targeting neurons from several cortical areas, including the prelimbic region (PL), anterior cingulate area (ACA) and secondary motor cortex (MOs). We found that each PPN-projecting neuron had a unique profile of collateralization, with some subcortical areas being preferential targets. In particular, PPN-projecting neurons from all three traced cortical areas exhibited common preferential collateralization to several nuclei, with most neurons targeting the striatum (STR), lateral hypothalamic area (LHA) and periaqueductal gray (PAG), and a substantial portion of neurons also targeting the zona incerta (ZI), median raphe nucleus (MRN) and substantia nigra pars reticulata (SNr). Meanwhile, very specific collateralization patterns were found for other nuclei, including the intermediate reticular nucleus (IRN), parvicellular reticular nucleus (PARN) and gigantocellular reticular nucleus (GRN), which receive collateral inputs almost exclusively from the MOs. These observations provide potential anatomical mechanisms for cortical neurons to coordinate the PPN with other subcortical areas in performing different physiological functions.

Single-cell reconstruction reveals input patterns and pathways into corticotropin-releasing factor neurons in the central amygdala in mice.

Corticotropin-releasing factor (CRF) neurons are one of the most densely distributed cell types in the central amygdala (CeA), and are involved in a wide range of behaviors including anxiety and learning. However, the fundamental input circuits and patterns of CeA-CRF neurons are still unclear. Here, we generate a monosynaptic-input map onto CeA-CRF neurons at single-cell resolution via a retrograde rabies-virus system. We find all inputs are located in 44 nested subregions that directly innervate CeA-CRF neurons; most of them are top-down convergent inputs expressing Ca2+/calmodulin-dependent protein kinase II, and are centralized in cortex, especially in the layer 4 of the somatosensory cortex, which may directly relay information from the thalamus. While the bottom-up divergent inputs have the highest proportion of glutamate decarboxylase expression. Finally, en passant structures of single input neuron are revealed by in-situ reconstruction in a modified 3D-reference atlas, represented by a Periaqueductal gray-Subparafascicular nucleus-Subthalamic nucleus-Globus pallidus-Caudoputamen-CeA pathway. Taken together, our findings provide morphological and connectivity properties of inputs onto CeA-CRF neurons, which may provide insights for future studies interrogating circuit mechanisms of CeA-CRF neurons in mediating various functions.

High-throughput mapping of a whole rhesus monkey brain at micrometer resolution.

Whole-brain mesoscale mapping in primates has been hindered by large brain sizes and the relatively low throughput of available microscopy methods. Here, we present an approach that combines primate-optimized tissue sectioning and clearing with ultrahigh-speed fluorescence microscopy implementing improved volumetric imaging with synchronized on-the-fly-scan and readout technique, and is capable of completing whole-brain imaging of a rhesus monkey at 1 × 1 × 2.5 µm3 voxel resolution within 100 h. We also developed a highly efficient method for long-range tracing of sparse axonal fibers in datasets numbering hundreds of terabytes. This pipeline, which we call serial sectioning and clearing, three-dimensional microscopy with semiautomated reconstruction and tracing (SMART), enables effective connectome-scale mapping of large primate brains. With SMART, we were able to construct a cortical projection map of the mediodorsal nucleus of the thalamus and identify distinct turning and routing patterns of individual axons in the cortical folds while approaching their arborization destinations.

Long-range GABAergic projections from the nucleus of the solitary tract.

The nucleus of the solitary tract (NTS) plays a crucial role in integrating peripheral information regarding visceral functions. Glutamate decarboxylase 2 (GAD2) inhibitory neurons are abundant in the NTS, and are known to form local and short-range projections within the NTS and nearby hindbrain areas. Here we performed whole-brain mapping of outputs from GAD2 neurons in the NTS using cell-type specific viral labeling together with ultrahigh-speed 3D imaging at 1-µm resolution. In addition to well-known targets of NTS GAD2 neurons including the principle sensory nucleus of the trigeminal (PSV), spinal nucleus of the trigeminal (SPV), and other short-range targets within the hindbrain, the high sensitivity of our system helps reveal previously unknown long-range projections that target forebrain regions, including the bed nuclei of the stria terminalis (BST) involved in stress and fear responses, and the paraventricular hypothalamic nucleus (PVH) involved in energy balance and stress-related neuroendocrine responses. The long-range projections were further verified by retrograde labeling of NTS GAD2 neurons with cholera toxin B (CTB) injections in the BST and PVH, and by Cre-dependent retrograde tracing with rAAV2-retro injections in the two regions of GAD2-Cre mice. Finally, we performed complete morphological reconstruction of several sparsely labeled neurons projecting to the forebrain and midbrain. These results provide new insights about how NTS might participate in physiological and emotional modulation.

Scalable volumetric imaging for ultrahigh-speed brain mapping at synaptic resolution.

The speed of high-resolution optical imaging has been a rate-limiting factor for meso-scale mapping of brain structures and functional circuits, which is of fundamental importance for neuroscience research. Here, we describe a new microscopy method of Volumetric Imaging with Synchronized on-the-fly-scan and Readout (VISoR) for high-throughput, high-quality brain mapping. Combining synchronized scanning beam illumination and oblique imaging over cleared tissue sections in smooth motion, the VISoR system effectively eliminates motion blur to obtain undistorted images. By continuously imaging moving samples without stopping, the system achieves high-speed 3D image acquisition of an entire mouse brain within 1.5 hours, at a resolution capable of visualizing synaptic spines. A pipeline is developed for sample preparation, imaging, 3D image reconstruction and quantification. Our approach is compatible with immunofluorescence methods, enabling flexible cell-type specific brain mapping and is readily scalable for large biological samples such as primate brains. Using this system, we examined behaviorally relevant whole-brain neuronal activation in 16 c-Fos-shEGFP mice under resting or forced swimming conditions. Our results indicate the involvement of multiple subcortical areas in stress response. Intriguingly, neuronal activation in these areas exhibits striking individual variability among different animals, suggesting the necessity of sufficient cohort size for such studies.

Corticosterone Signaling and a Lateral Habenula-Ventral Tegmental Area Circuit Modulate Compulsive Self-Injurious Behavior in a Rat Model.

Self-injurious behavior (SIB) is commonly observed in patients with neuropsychiatric disorders, as well as in nonclinical populations with stress-related mental-health problems. However, the exact circuitry mechanisms underlying SIB have remained poorly understood. Here, with bilateral injection of muscimol into the entopeduncular nucleus (EP), we established a rat model of SIB. Following the muscimol injection, the male rats exhibited in a dose-dependent manner stereotypic self-biting behavior that lasted for hours and often resulted in wounds of various severities. The SIB was associated with an elevated level of serum corticosterone and could be exacerbated by enhancing the corticosterone signaling and, conversely, alleviated by inhibiting the corticosterone signaling. Activity mapping using c-fos immunostaining, combined with connectivity mapping using herpes simplex virus-based anterograde tracing from the EP and pseudorabies virus-based retrograde tracing from the masseter muscle, revealed the potential involvement of many brain areas in SIB. In particular, the lateral habenula (LHb) and the ventral tegmental area (VTA), the two connected brain areas involved in stress response and reward processing, showed a significant increase in neuronal activation during SIB. Furthermore, suppressing the LHb activity or modulating the GABAergic transmission in the VTA could significantly reduce the occurrence of SIB. These results demonstrate the importance of stress hormone signaling and the LHb-VTA circuit in modulating SIB resulting from EP malfunction, and suggest potential targets for therapeutic intervention of SIB and related disorders.SIGNIFICANCE STATEMENT Self-injurious behavior (SIB) occurs in ∼4% of the general population, with substantially higher occurrence among adolescents and patients of neuropsychiatric disorders. Stress has been linked to the occurrence of SIB, yet the underlying mechanisms have remained unclear. Using a rat model of SIB induced by disruption of activity in the entopeduncular nucleus (EP), we found that the behavior is regulated by stress and linked to corticosterone signaling. Viral tracing and c-fos immunostaining revealed the involvement of various subcortical areas, especially the EP-lateral habenula (LHb)-ventral tegmental area (VTA) circuit, in SIB. Furthermore, regulating activity in the LHb or the VTA alleviates SIB. These results may have implications in the development of new strategies for treating SIB.