Brain microinfarcts are prevalent in humans, however due to the inherent difficulty of identifying and localizing individual microinfarcts, brain-wide quantification is impractical. In mice, microinfarcts have been created by surgically introducing microemboli into the brain, but a major limitation of this model is the absence of automated methods to identify and localize individual occlusions. We present a novel and semi-automated workflow to identify the anatomical location of fluorescent emboli (microspheres) within the mouse brain through histological processing and atlas registration. By incorporating vibratome block-face imaging with the QuickNII brain registration tool, we show that the anatomical location of microspheres can be accurately registered to brain structures within the Allen mouse brain (AMB) atlas (e.g. somatomotor areas, hippocampal region, visual areas, etc.). Compared to registering images of slide mounted sections to the AMB atlas, microsphere location was more accurately determined...

Adaptive reward-related decision making requires accurate prospective consideration of the specific outcome of each option and its current desirability. Often this information must be inferred based on the presence of predictive environmental events. The basolateral amygdala (BLA) and medial orbitofrontal cortex (mOFC) are two key nodes in the circuitry supporting such outcome expectations, but very little is known about the function of direct connections between these regions. Here, in male rats, we first anatomically confirmed the existence of bidirectional, direct projections between the mOFC and BLA and found that BLA projections to mOFC are largely distinct from those to lateral OFC (lOFC). Next, using pathway-specific chemogenetic inhibition and the outcome-selective Pavlovian-to-instrumental transfer and devaluation tests, we interrogated the function of the bidirectional mOFC-BLA connections in reward-directed behavior. We found evidence that the mOFC→BLA pathway mediates the use of environmental c...

Comorbid anxiety and depressive symptoms in chronic pain are a common health problem, but the underlying mechanisms remain unclear. Previously, we have demonstrated that sensitization of the central nucleus of the amygdala (CeA) neurons via decreased GABAergic inhibition contributes to anxiety-like behaviors in neuropathic pain rats. In this study, by using male Sprague-Dawley rats, we reported that the CeA plays a key role in processing both sensory and negative emotional-affective components of neuropathic pain. Bilateral electrolytic lesions of CeA but not lateral/basolateral nucleus of the amygdala (LA/BLA) abrogated both pain hypersensitivity and aversive and depressive symptoms of neuropathic rats induced by spinal nerve ligation (SNL). Moreover, SNL rats showed structural and functional neuroplasticity manifested as reduced dendritic spines on the CeA neurons and enhanced long-term depression (LTD) at the LA/BLA-CeA synapse. Disruption of GluA2-containing AMPARs trafficking and endocytosis from syna...

Iron is an essential co-factor for several metabolic processes, including the generation of adenosine triphosphate (ATP) in mitochondria, which is required for axonal function and regeneration. However, it is not known how mitochondria in long axons, such as those in sciatic nerves, acquire iron in vivo . Due to their close proximity to axons, Schwann cells (SCs) are a likely source of iron for axonal mitochondria in the peripheral nervous system. Here we demonstrate the critical role of iron in promoting neurite growth in vitro using iron chelation. We also show that SCs express the molecular machinery to release iron, namely, the iron exporter, ferroportin (Fpn) and the ferroxidase ceruloplasmin (Cp). In Cp knockout (KO) mice, SCs accumulate iron, because Fpn requires to partner with Cp to export iron. Axons and SCs also express the iron importer transferrin receptor 1 (TfR1), indicating their ability for iron uptake. In teased nerve fibers, Fpn and TfR1 are predominantly localized at the nodes of Ranvie...

Mutations in the X-linked cell adhesion protein PCDH19 lead to seizures, cognitive impairment and other behavioral comorbidities when present in a mosaic pattern. Neither the molecular mechanisms underpinning this disorder, nor the function of PCDH19 itself are well understood. By combining RNA in situ hybridization with immunohistochemistry and analyzing single cell RNAseq datasets, we reveal Pcdh19 expression in cortical interneurons and provide a first account of the subtypes of neurons expressing Pcdh19 / PCDH19 , both in the mouse and the human cortex. Our quantitative analysis of the Pcdh19 mutant mouse exposes subtle changes in cortical layer composition, with no major alterations of the main axonal tracts. In addition, Pcdh19 mutant animals, particularly females, display preweaning behavioral changes, including reduced anxiety and increased exploratory behavior. Importantly, our experiments also reveal an effect of the social environment on the behavior of wild-type littermates of Pcdh19 mutant mic...

The enteric nervous system (ENS) consists of an interconnected meshwork of neurons and glia residing within the wall of the gastrointestinal (GI) tract. While healthy GI function is associated with healthy ENS structure, defined by the normal distribution of neurons within ganglia of the ENS, a comprehensive understanding of normal neuronal distribution and ganglionic organization in the ENS is lacking. Current methodologies for manual enumeration of neurons parse only limited tissue regions and are prone to error, subjective bias, and peer-to-peer discordance. There is accordingly a need for robust, and objective tools that can capture and quantify enteric neurons within multiple ganglia over large areas of tissue. Here, we report on the development of an AI-driven tool, COUNTEN (COUNTing Enteric Neurons), which is capable of accurately identifying and enumerating immunolabeled enteric neurons, and objectively clustering them into ganglia. We tested and found that COUNTEN matches trained humans in its acc...

The N-methyl-D-aspartate (NMDA) receptor is a type of glutamate receptor, which is involved in neuronal function, plasticity and development in the mammalian brain. However, how the NMDA receptors contribute to adult neurogenesis and development of the dentate gyrus is unclear. In this study, we investigate this question by examining a region-specific knockout mouse line that lacks the NR1 gene, which encodes the essential subunit of the NMDA receptors, in granule cells of the dentate gyrus (DG-NR1 KO mice). We found that the survival of newly-generated granule cells, cell proliferation and the size of the granule cell layer are significantly reduced in the dorsal dentate gyrus of adult DG-NR1 KO mice. Our results also show a significant reduction in the number of immature neurons and in the volume of the granule cell layer, starting from 3 weeks of postnatal age. DG-NR1KO mice also showed impairment in the expression of an immediate early gene, Arc, and behavior during the novelty-suppressed feeding and o...

Activity-dependent insertion of the tropomyosin-related kinase B (TrkB) receptor into the plasma membrane can explain, in part, the preferential effect of brain-derived neurotrophic factor (BDNF) on active neurons and synapses; however, the underlying molecular mechanisms remain obscure. Here, we report a novel function for carboxypeptidase E (CPE) in controlling chemical long-term potentiation (cLTP) stimuli-induced TrkB surface delivery in hippocampal neurons. Total internal reflection fluorescence (TIRF) assays and line plot assays showed that CPE facilitates TrkB transport from dendritic shafts to the plasma membrane. The Box2 domain in the juxtamembrane region of TrkB and the C-terminus of CPE are critical for the activity-dependent plasma membrane insertion of TrkB. Moreover, TAT-CPE452-466, which could block the association between CPE and TrkB, significantly inhibited neuronal activity-enhanced BDNF signaling and dendritic spine morphological plasticity in cultured hippocampal neurons. Microinfusio...

Structural plasticity of dendritic spines is considered to be an important basis of synaptic plasticity, learning and memory. Here, we induced input-specific structural LTP (sLTP) in single dendritic spines in organotypic hippocampal slices from mice of either sex and performed ultrastructural analyses of the spines using efficient correlative light and electron microscopy (CLEM). We observed reorganization of the PSD nanostructure, such as perforation and segmentation, at 2-3, 20, and 120 min after sLTP induction. In addition, PSD and nonsynaptic axon-spine interface (nsASI) membrane expanded unevenly during sLTP. Specifically, the PSD area showed a rapid increase at 2-3 min after sLTP induction. The PSD growth was to a degree less than spine volume growth at 2-3 min and 20 min after sLTP induction but became similar at 120 min. On the other hand, the nsASI area showed a profound and lasting expansion, to a degree similar to spine volume growth throughout the process. These rapid ultrastructural changes i...

Evidence from animal and human research shows that established memories can undergo changes after reactivation through a process called reconsolidation. Alterations of the level of the stress hormone cortisol may provide a way to manipulate reconsolidation in humans. Here, in a double-blind, within-subject design, we reactivated a 3-day-old memory at 3:55 a.m. in sixteen men and four women, immediately followed by oral administration of metyrapone vs. placebo, to examine whether metyrapone-induced suppression of the morning cortisol rise may influence reconsolidation processes during and after early morning sleep. Crucially, reactivation followed by cortisol suppression vs. placebo resulted in enhanced memory for the reactivated episode tested four days after reactivation. This enhancement after cortisol suppression was specific for the reactivated episode vs. a non-reactivated episode. These findings suggest that when reactivation of memories is immediately followed by suppression of cortisol levels durin...