Retrograde Tracing Research Articles

PACAP Controls Endocrine and Behavioral Stress Responses via Separate Brain Circuits

BackgroundThe neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) is a master regulator of central and peripheral stress responses, yet it is not clear how PACAP projections throughout the brain execute endocrine and behavioral stress responses. MethodsWe used AAV (adeno-associated virus) neuronal tracing, an acute restraint stress (ARS) paradigm, and intersectional genetics, in C57BL/6 mice, to identify PACAP-containing circuits controlling stress-induced behavior and endocrine activation. ResultsPACAP deletion from forebrain excitatory neurons, including a projection directly from medial prefrontal cortex to hypothalamus, impairs c-fos activation and corticotropin-releasing hormone (CRH) messenger RNA elevation in the paraventricular nucleus after 2 hours of restraint, without affecting ARS-induced hypophagia, or c-fos elevation in nonhypothalamic brain. Elimination of PACAP within projections from lateral parabrachial nucleus to extended amygdala, on the other hand, attenuates ARS-induced hypophagia, along with extended amygdala fos induction, without affecting ARS-induced CRH messenger RNA elevation in the paraventricular nucleus. PACAP projections to extended amygdala terminate at protein kinase C delta type (PKCδ) neurons in both the central amygdala and the oval bed nucleus of the stria terminalis. Silencing of PKCδ neurons in the central amygdala, but not in the oval bed nucleus of the stria terminalis, attenuates ARS-induced hypophagia. Experiments were carried out in mice of both sexes with n ≥ 3 per group. ConclusionsA frontocortical descending PACAP projection controls paraventricular nucleus CRH messenger RNA production to maintain hypothalamic-pituitary-adrenal axis activation and regulate the endocrine response to stress. An ascending PACAPergic projection from the external lateral parabrachial nucleus to PKCδ neurons in the central amygdala regulates behavioral responses to stress. Defining two separate limbs of the acute stress response provides broader insight into the specific brain circuitry engaged by the psychogenic stress response.

Nos1+ and Nos1− excitatory neurons in the BLA regulate anxiety- and depression-related behaviors oppositely

BackgroundThe basolateral amygdala (BLA) neurons are primarily glutamatergic and have been associated with emotion regulation. However, little is known about the roles of BLA neurons expressing neuronal nitric oxide synthase (nNOS, Nos1) in the regulation of emotional behaviors. MethodsUsing Nos1−cre mice and chemogenetic and optogenetic manipulations, we specifically silenced or activated Nos1+ or Nos1− neurons in the BLA, or silenced their projections to the anterdorsal bed nucleus of the stria terminalis (adBNST) and ventral hippocampus (vHPC). We measured anxiety behaviors in elevated plus maze (EPM) and open-field test (OFT), and measured depression behaviors in forced swimming test (FST) and tail suspension test (TST). ResultsBLA Nos1+ neurons were predominantly glutamatergic, and glutamatergic but not GABAergic Nos1+ neurons were involved in controlling anxiety- and depression-related behaviors. Interestingly, by selectively manipulating the activities of BLA Nos1+ and Nos1− excitatory neurons, we found that they had opposing effects on anxiety- and depression-related behaviors. BLA Nos1+ excitatory neurons projected to the adBNST, this BLA-adBNST circuit controlled the expression of anxiety- and depression-related behaviors, while BLA Nos1− excitatory neurons projected to vHPC, this BLA-vHPC circuit contributed to the expression of anxiety- and depression-related behaviors. Moreover, excitatory vHPC-adBNST circuit antagonized the role of BLA-adBNST circuit in regulating anxiety- and depression-related behaviors. ConclusionsBLA Nos1+ and Nos1− excitatory neuron subpopulations exert different effects on anxiety- and depression-related behaviors through distinct projection circuits, providing a new insight of BLA excitatory neurons in emotional regulation. LimitationsWe did not perform retrograde labeling from adBNST and vHPC regions.

BigNeuron: a resource to benchmark and predict performance of algorithms for automated tracing of neurons in light microscopy datasets.

BigNeuron is an open community bench-testing platform with the goal of setting open standards for accurate and fast automatic neuron tracing. We gathered a diverse set of image volumes across several species that is representative of the data obtained in many neuroscience laboratories interested in neuron tracing. Here, we report generated gold standard manual annotations for a subset of the available imaging datasets and quantified tracing quality for 35 automatic tracing algorithms. The goal of generating such a hand-curated diverse dataset is to advance the development of tracing algorithms and enable generalizable benchmarking. Together with image quality features, we pooled the data in an interactive web application that enables users and developers to perform principal component analysis, t-distributed stochastic neighbor embedding, correlation and clustering, visualization of imaging and tracing data, and benchmarking of automatic tracing algorithms in user-defined data subsets. The image quality metrics explain most of the variance in the data, followed by neuromorphological features related to neuron size. We observed that diverse algorithms can provide complementary information to obtain accurate results and developed a method to iteratively combine methods and generate consensus reconstructions. The consensus trees obtained provide estimates of the neuron structure ground truth that typically outperform single algorithms in noisy datasets. However, specific algorithms may outperform the consensus tree strategy in specific imaging conditions. Finally, to aid users in predicting the most accurate automatic tracing results without manual annotations for comparison, we used support vector machine regression to predict reconstruction quality given an image volume and a set of automatic tracings.

Beneficial Effect of Bee Venom and Its Major Components on Facial Nerve Injury Induced in Mice.

Peripheral nerve injury (PNI) is a health problem that affects many people worldwide. This study is the first to evaluate the potential effect of bee venom (BV) and its major components in a model of PNI in the mouse. For that, the BV used in this study was analyzed using UHPLC. All animals underwent a distal section-suture of facial nerve branches, and they were randomly divided into five groups. Group 1: injured facial nerve branches without any treatment. Group 2: the facial nerve branches were injured, and the normal saline was injected similarly as in the BV-treated group. Group 3: injured facial nerve branches with local injections of BV solution. Group 4: injured facial nerve branches with local injections of a mixture of PLA2 and melittin. Group 5: injured facial nerve branches with local injection of betamethasone. The treatment was performed three times a week for 4 weeks. The animals were submitted to functional analysis (observation of whisker movement and quantification of nasal deviation). The vibrissae muscle re-innervation was evaluated by retrograde labeling of facial motoneurons in all experimental groups. UHPLC data showed 76.90 ± 0.13%, 11.73 ± 0.13%, and 2.01 ± 0.01%, respectively, for melittin, phospholipase A2, and apamin in the studied BV sample. The obtained results showed that BV treatment was more potent than the mixture of PLA2 and melittin or betamethasone in behavioral recovery. The whisker movement occurred faster in BV-treated mice than in the other groups, with a complete disappearance of nasal deviation two weeks after surgery. Morphologically, a normal fluorogold labeling of the facial motoneurons was restored 4 weeks after surgery in the BV-treated group, but no such restoration was ever observed in other groups. Our findings indicate the potential of the use of BV injections to enhance appropriate functional and neuronal outcomes after PNI.

Compensatory remodeling of a septo-hippocampal GABAergic network in the triple transgenic Alzheimer’s mouse model

BackgroundAlzheimer’s disease (AD) is characterized by a progressive loss of memory that cannot be efficiently managed by currently available AD therapeutics. So far, most treatments for AD that have the potential to improve memory target neural circuits to protect their integrity. However, the vulnerable neural circuits and their dynamic remodeling during AD progression remain largely undefined.MethodsCircuit-based approaches, including anterograde and retrograde tracing, slice electrophysiology, and fiber photometry, were used to investigate the dynamic structural and functional remodeling of a GABAergic circuit projected from the medial septum (MS) to the dentate gyrus (DG) in 3xTg-AD mice during AD progression.ResultsWe identified a long-distance GABAergic circuit that couples highly connected MS and DG GABAergic neurons during spatial memory encoding. Furthermore, we found hyperactivity of DG interneurons during early AD, which persisted into late AD stages. Interestingly, MS GABAergic projections developed a series of adaptive strategies to combat DG interneuron hyperactivity. During early-stage AD, MS-DG GABAergic projections exhibit increased inhibitory synaptic strength onto DG interneurons to inhibit their activities. During late-stage AD, MS-DG GABAergic projections form higher anatomical connectivity with DG interneurons and exhibit aberrant outgrowth to increase the inhibition onto DG interneurons.ConclusionWe report the structural and functional remodeling of the MS-DG GABAergic circuit during disease progression in 3xTg-AD mice. Dynamic MS-DG GABAergic circuit remodeling represents a compensatory mechanism to combat DG interneuron hyperactivity induced by reduced GABA transmission.

Involvement of cortical projections to basolateral amygdala in context-induced reinstatement of ethanol-seeking in rats

Ethanol is the most consumed substance of abuse in the world, and its misuse may lead to the development of alcohol use disorder (AUD). High relapse rates remain a relevant problem in the treatment of AUD. Exposure to environmental cues previously associated with ethanol intake could trigger ethanol-seeking behavior. However, the neural mechanisms involved in this phenomenon are not entirely clear. In this context, cortical projections to the basolateral amygdala (BLA) play a role in appetitive and aversive learned behaviors. Therefore, we aimed to evaluate the activation of the cortical projections from the prelimbic (PL), orbitofrontal (OFC), and infralimbic (IL), to the BLA in the context-induced reinstatement of ethanol-seeking. Male Long-Evans rats were trained to self-administer 10% ethanol in Context A. Subsequently, lever pressing in the presence of the discrete cue was extinguished in Context B. After nine extinction sessions, rats underwent intracranial surgery for the unilateral injection of red fluorescent retrograde tracer into the BLA. The context-induced reinstatement of ethanol-seeking was assessed by re-exposing the rats to Context A or B under extinction conditions. Finally, we combined retrograde neuronal tracing with Fos to identify activated cortical inputs to BLA during the reinstatement of ethanol-seeking behavior. We found that PL, but not OFC or IL, retrogradely-labeled neurons from BLA presented increased Fos expression during the re-exposure to the ethanol-associated context, suggesting that PL projection to BLA is involved in the context-induced reinstatement of ethanol-seeking behavior.

Perineurial Window is Critical for Experimental Reverse End-to-Side Nerve Transfer.

The depth of connective tissue window in the side of a recipient nerve in reverse end-to-side transfers (RETS) remains controversial. To test whether the depth of connective tissue disruption influences the efficiency of donor axonal regeneration in the context of RETS. Sprague-Dawley rats (n = 24) were assigned to 1 of the 3 groups for obturator nerve to motor femoral nerve RETS: group 1, without epineurium opening; group 2, with epineurium only opening; and group 3, with epineurium and perineurium opening. Triple retrograde labeling was used to assess the number of motor neurons that had regenerated into the recipient motor femoral branch. Thy1-GFP rats (n = 8) were also used to visualize the regeneration pathways in the nerve transfer networks at 2- and 8-week time point using light sheet fluorescence microscopy. The number of retrogradely labeled motor neurons that had regenerated distally toward the target muscle was significantly higher in group 3 than that in groups 1 and 2. Immunohistochemistry validated the degree of connective tissue disruption among the 3 groups, and optical tissue clearing methods demonstrated donor axons traveling outside the fascicles in groups 1 and 2 but mostly within the fascicles in group 3. Creating a perineurial window in the side of recipient nerves provides the best chances of robust donor axonal regeneration across the RETS repair site. This finding aids nerve surgeons by confirming that a deep window should be undertaken when doing a RETS procedure.

Molecular Profiling of VGluT1 AND VGluT2 Ventral Subiculum to Nucleus Accumbens Shell Projections.

The nucleus accumbens shell is a critical node in reward circuitry, encoding environments associated with reward. Long-range inputs from the ventral hippocampus (ventral subiculum) to the nucleus accumbens shell have been identified, yet their precise molecular phenotype remains to be determined. Here we used retrograde tracing to identify the ventral subiculum as the brain region with the densest glutamatergic (VGluT1-Slc17a7) input to the shell. We then used circuit-directed translating ribosome affinity purification to examine the molecular characteristics of distinct glutamatergic (VGluT1, VGluT2-Slc17a6) ventral subiculum to nucleus accumbens shell projections. We immunoprecipitated translating ribosomes from this population of projection neurons and analysed molecular connectomic information using RNA sequencing. We found differential gene enrichment across both glutamatergic projection neuron subtypes. In VGluT1 projections, we found enrichment of Pfkl, a gene involved in glucose metabolism. In VGluT2 projections, we found a depletion of Sparcl1 and Dlg1, genes known to play a role in depression- and addiction-related behaviours. These findings highlight potential glutamatergic neuronal-projection-specific differences in ventral subiculum to nucleus accumbens shell projections. Together these data advance our understanding of the phenotype of a defined brain circuit.

C-type natriuretic peptide (CNP) in the paraventricular nucleus-mediated renal sympatho-inhibition.

Volume reflex produces sympatho-inhibition that is mediated by the hypothalamic paraventricular nucleus (PVN). However, the mechanisms for the sympatho-inhibitory role of the PVN and the neurochemical factors involved remain to be identified. In this study, we proposed C-type natriuretic peptide (CNP) as a potential mediator of this sympatho-inhibition within the PVN. Microinjection of CNP (1.0μg) into the PVN significantly decreased renal sympathetic nerve activity (RSNA) (-25.8% ± 1.8% vs. -3.6% ± 1.5%), mean arterial pressure (-15.0 ± 1.9 vs. -0.1 ± 0.9mmHg) and heart rate (-23.6 ± 3.5 vs. -0.3 ± 0.9 beats/min) compared with microinjection of vehicle. Picoinjection of CNP significantly decreased the basal discharge of extracellular single-unit recordings in 5/6 (83%) rostral ventrolateral medulla (RVLM)-projecting PVN neurons and in 6/13 (46%) of the neurons that were not antidromically activated from the RVLM. We also observed that natriuretic peptide receptor type C (NPR-C) was present on the RVLM projecting PVN neurons detected by dual-labeling with retrograde tracer. Prior NPR-C siRNA microinjection into the PVN significantly blunted the decrease in RSNA to CNP microinjections into the PVN. Volume expansion-mediated reduction in RSNA was significantly blunted by prior administration of NPR-C siRNA into the PVN. These results suggest a potential role for CNP within the PVN in regulating RSNA, specifically under physiological conditions of alterations in fluid balance.

Abstract 1193: Sensory innervation directly promotes gastric cancer and modulates the tumor microenvironment

Abstract Tumor innervation contributes directly to cancer progression, but there appears to be organ-specific variation in the type of innervation. While our previous studies showed cholinergic signaling promoted gastric cancer and vagal denervation inhibited gastric cancer, it is noteworthy that sensory nerves comprise 80% of the vagal axons. To investigate the role of sensory innervation in gastric carcinogenesis, we performed retrograde neural tracing and identified CGRP+ peptidergic neurons in nodose ganglia and dorsal root ganglia as primary gastric sensory neurons. While VAChT+ parasympathetic nerves are the main nerves innervating the normal stomach, CGRP+ sensory nerves show the greatest degree of expansion in mouse gastric tumors. Indeed, mouse gastric tumors show upregulation of NGF, a growth factor essential for sensory nerve survival, and gastric NGF overexpression in an NGF knock-in mouse also resulted in increased sensory innervation. Inhibition of NGF/Trk signaling by Entrectinib markedly attenuates NGF-dependent sensory nerve expansion. In addition to overall increased axonogenesis of sensory nerves, mouse models of gastric cancer showed upregulation of αCGRP peptide in gastric tumors and Calca mRNA in gastric sensory neurons, indicating increased activation of sensory neurons during gastric carcinogenesis. To study the effect of such activation, sensory neurons were then directly activated by treatment of Trpv1-Cre;hM3Dq mice with clozapine-N-oxide (CNO), which led to marked gastric hyperplasia with increased proliferation. Interestingly, in an acetic acid-induced ulcer model, activation of sensory neurons reduced the ulcer size and strongly promoted gland regeneration. Activation of sensory neurons, in a syngeneic, orthotopic model of gastric cancer in the Trpv1-Cre;hM3Dq mice, led to a significant increase in tumor volume, Ki-67+ cancer cells, CD31+ vessels and M2 macrophage polarization, as well as decreased CD8+ T cells. Analysis of available single-cell transcriptome data from mouse and human stomach showed that Calcrl/Ramp1 (CGRP receptors) are expressed in gastric epithelial cells and most stromal cells, while the expression levels of Tacr1 (receptor for Substance P) and Sstr1-5 (receptors for Somatostatin) were much lower. Importantly, some Calcrl/Ramp1+ cells are proliferative cells with strong Ki-67 expression. Our data show that Calcrl is also overexpressed in mouse gastric tumors, while TCGA data reveals significant upregulation of CALCRL in human gastric cancers and that high expression of CALCRL is correlated with poor overall survival. Finally, we established a co-culture model using DRGs and gastric cancer organoids, and found that sensory nerves can generate synaptic-like terminals on cancer organoids. In conclusion, our data indicate that sensory innervation directly promotes gastric cancer growth and also modulates the tumor microenvironment. Citation Format: Xiaofei Zhi, Feijing Wu, Jin Qian, Yosuke Ochiai, Guodong Lian, Ermanno Malagola, Timothy C. Wang. Sensory innervation directly promotes gastric cancer and modulates the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1193.

Abstract 3630: TrkA+ sensory neurons modulate macrophage phenotype in osteosarcoma

Abstract Introduction: Osteosarcoma (OS) is the most common primary malignancy of the skeleton, and inordinately affects young adults. Despite improved outcomes with current multimodal care, the prognosis for patients with metastatic disease is grim. Peripheral neural regulation of cancer growth has long been suggested by clinical observations in pancreatic and prostate cancers. Prior reports suggest peripheral nerve sprouting in osteosarcoma, but the functional roles of sensory nerves in osteosarcoma are unknown. Here, the role of TrkA+ sensory nerves in OS disease progression was assayed. Methods: All experiments were conducted under IACUC approval at Johns Hopkins University. TrkAF592A mice which are homozygous for a phenylalanine-to-alanine point mutation in exon 12 of Ntrk1 gene were crossbred with NOD scid mice (TrkAF592A/Scid). TrkA inhibition was performed by 1NMPP1 treatment. In vivo tumor growth was examined using a periosteal xenograft model. 1 million human 143B-luc OS cells were injected. Analysis was performed using serial measurements of tumor size and IVIS imaging. Nerve distribution and macrophage polarization within the tumor microenvironment was documented using immunostaining for Beta III Tubulin (TUBB3), CD86, CD206 and F4/80. Results: Inhibition of TrkA led to a significant reduction in Tubb3+ nerves (88.4%) within and around the OS xenografts. In addition, tumors within TrkAF592A/Scid animals demonstrated slower growth, including a 51.5% reduction in tumor size and 60.0% reduction in IVIS luciferase activity at 4 wks. Histologic examination showed a reduction in macrophage infiltration and reprogramming of macrophages from an M2-like to M1-like phenotype in tumors with chemical denervation. Remarkably, this was associated with a reduction in Ki67 proliferative index (80.1%) and CD31 immunofluorescent staining (60.8%) within the tumors. Overall survival of mice with TrkA inhibition was significantly prolonged (p=0.0003). Retrograde tracing identified the sensory nerve fibers innervating the periosteum originated from L3-L5 lumbar DRGs and RNA seq identified OS-derived neurotrophins and axon guidance molecules. Discussion: Disruption of TrkA+ nerves significantly mitigates OS disease progression in a mouse xenograft model, including a significant improved overall survival. These findings are associated with a reduction of macrophage infiltration and macrophage re-polarization. Ongoing studies are examining the molecular mechanisms that mediate nerve-to-macrophage interaction in peripheral nerves and DRG neurons using a combination of sequencing approaches. Significance/Clinical Relevance: Sensory neurons positively regulate osteosarcoma growth and disease progression. Use of chemical or microsurgical approaches to denervate tumors may represent a future adjunctive therapy to both improve pain and prognosis in high grade bone sarcomas. Citation Format: Qizhi Qin, Sowmya Ramesh Ramesh, Thomas L. Clemens, Aaron W. James. TrkA+ sensory neurons modulate macrophage phenotype in osteosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3630.

Sensory innervation of the lumbar 5/6 intervertebral disk in mice.

Over the years, most back pain-related biological studies focused on the pathogenesis of disk degeneration. It is known that nerve distributions at the outer layer of the annulus fibrosus (AF) may be an important contributor to back pain symptoms. However, the types and origins of sensory nerve terminals in the mouse lumbar disks have not been widely studied. Using disk microinjection and nerve retrograde tracing methods, the current study aimed to characterize the nerve types and neuropathway of the lumbar 5/6 (L5/6) disk in mice. Using an anterior peritoneal approach, the L5/6 disk of adult C57BL/6 mice (males, 8-12 weeks) disk microinjection was performed. Fluorogold (FG) was injected into the L5/6 disk using the Hamilton syringe with a homemade glass needle driven by a pressure microinjector. The lumbar spine and bilateral thoracic 13 (Th13) to L6 DRGs were harvested at 10 days after injection. The number of FG+ neurons among different levels was counted and analyzed. Different nerve markers, including anti-neurofilament 160/200 (NF160/200), anti-calcitonin gene-related peptide (CGRP), anti-parvalbumin (PV), and anti-tyrosine hydroxylase (TH), were used to identify different types of nerve terminals in AF and their origins in DRG neurons. There were at least three types of nerve terminals at the outer layer of L5/6 AF in mice, including NF160/200+ (indicating Aβ fibers), CGRP+ (Aδ and C fibers), and PV+ (proprioceptive fibers). No TH+ fibers (sympathetic nerve fibers and some C-low threshold mechanoreceptors) were noticed in either. Using retrograde tracing methods, we found that nerve terminals in the L5/6 disk were multi-segmentally from Th13-L6 DRGs, with L1 and L5 predominately. An immunofluorescence analysis revealed that FG+ neurons in DRGs were co-localized with NF160/200, CGRP, and PV, but not TH. Intervertebral disks were innervated by multiple types of nerve fibers in mice, including Aβ, Aδ, C, and proprioceptive fibers. No sympathetic nerve fibers were found in AF. The nerve network of the L5/6 disk in mice was multi-segmentally innervated by the Th13-L6 DRGs (mainly L1 and L5 DRGs). Our results may serve as a reference for preclinical studies of discogenic pain in mice.

Analysis of the spinal and vagal afferent innervation of the mouse colon using neuronal retrograde tracers.

The gut-brain axis has received increasing attention recently due to evidence that colonic microbes can affect brain function and behavior. However, little is known about the innervation of the colon by a major component of the gut-brain axis, vagal afferent neurons. Furthermore, it is currently unknown whether individual NG neurons or DRG neurons innervate both the proximal and distal colon. We aimed to quantify the number of vagal and spinal afferent neurons that innervate the colon; and determine whether these individual neurons simultaneously innervate the mouse proximal and distal colon. C57Bl/6 mice received injections of a combination of retrograde tracers that were either injected into the muscularis externa of the proximal or the distal colon: fast blue, DiI and DiO. Five to seven percent of lumbosacral and thoracolumbar spinal afferent neurons, and 25% of vagal afferent neurons were labelled by injections of DiI and DiO into the colon. We also found that approximately 8% of NG neurons innervate the distal colon. Ten percent of labeled thoracolumbar and 15% of labeled lumbosacral DRG neurons innervate both the distal and proximal colon. Eighteen percent of labeled NG neurons innervated both the distal and proximal colon. In conclusion, vagal afferent innervation of the distal colon is less extensive than the proximal colon, whereas a similar gradient was not observed for the spinal afferent innervation. Furthermore, overlap appears to exist between the receptive fields of vagal and spinal afferent neurons that innervate the proximal and distal colon.

Ten-m4 plays a unique role in the establishment of binocular visual circuits.

The patterning of binocular vision requires distinct molecular pathways for inputs arising from each side of the nervous system. Recent studies have demonstrated important roles for members of the Ten-m/Odz/teneurin family in the development of ipsilateral retinal projections. Here, we further highlight the significance of this gene family in visual development by identifying a role for Ten-m4 during the formation of the ipsilateral projection in the mouse. Ten-m4 was found to be expressed in the retina, dorsal lateral geniculate nucleus (dLGN), superior colliculus (SC), and primary visual cortex (V1) during development. Anterograde and retrograde tracing experiments in Ten-m4 knockout (KO) mice revealed a specific increase in ipsilateral retinal ganglion cells projecting to dLGN and SC. This increase was most prominent in regions corresponding to temporal retina. Consistent with this, EphB1 expression in the retina around the time of decussation was enhanced in this temporal region for KO mice, suggesting that the increased size of the ipsilateral population arises due to an increased number of retinal ganglion cells remaining ipsilaterally at the optic chiasm due to EphB1-mediated repulsion. The ectopic ipsilaterally targeted retinal ganglion cell projection observed in Ten-m4 KOs was associated with changes in response to ethologically-relevant visual stimuli. Together, these data demonstrate a requirement for Ten-m4 in the establishment of ipsilateral projections from the retina, which likely acts in combination with other Ten-m members (Ten-m2 and Ten-m3) to promote the formation of functional binocular circuits. This article is protected by copyright. All rights reserved.

Divergent projections from locus coeruleus to the corticobasal ganglia system and ventral tegmental area of the adult male zebra finch.

The locus coeruleus (LC) is a small noradrenergic brainstem nucleus that plays a central role in regulating arousal, attention, and performance. In the mammalian brain, individual LC neurons make divergent axonal projections to different brain regions, which are distinguished in part by which noradrenaline (NA) receptor subtypes they express. Here, we sought to determine whether similar organizational features characterize LC projections to corticobasal ganglia (CBG) circuitry in the zebra finch song system, with a focus on the basal ganglia nucleus Area X, the thalamic nucleus DLM, as well as the cortical nuclei HVC, LMAN, and RA. Single and dual retrograde tracer injections reveal that single LC-NA neurons make divergent projections to LMAN and Area X, as well as to the dopaminergic VTA/SNc complex that innervates this CBG circuit. Moreover, in situ hybridization revealed that differential expression of mRNA encoding α2A and α2C adrenoreceptors distinguishes LC-recipient CBG song nuclei. Therefore, LC-NA signaling in the zebra finch CBG circuit employs a similar strategy as in mammals, which could allow a relatively small number of LC neurons to exert widespread yet distinct effects across multiple brain regions.

Morphological analysis of neck muscle nerves and neurons in cats

Previous studies have failed to show morphological differences between neck muscle alpha and gamma motor fibers or alpha and gamma motoneurons. The present study aimed to investigate the morphological features of neck muscle motor nerves and motoneurons in cats. To determine the morphological features of peripheral motor fibers, the value of the outer contours of each fiber was converted into a perfect circle after ganglionectomy to remove sensory fibers, and the fiber diameters were calculated based on their circumferences. The sizes of neck motor fibers in the peripheral nerves had an evident bimodal distribution into small and large fiber groups, as depicted in histograms. The sizes of small and large motor fibers ranged from 2 to 12 µm and from 12 to 40 µm, respectively. The small fiber group is likely to correspond to gamma motor fibers and the large fiber group to alpha motor fibers. The morphological features of neck muscle motoneurons sectioned in the horizontal plane were examined using the horseradish peroxidase (HRP) retrograde labeling technique. The diameters of the biventer cervicis and complexus motoneurons had bimodal distributions. The inflection point between the small and large diameter population was 28 µm for the biventer cervicis and 26 µm for the complexus. We also observed that larger neurons displayed more dendrites. In conclusion, we could identify morphological differences likely to correlate with alpha and gamma motoneurons in both neck muscle peripheral nerves and neck motoneurons.

Serotonin is a gap junction-permeable neuronal tracer in the mouse retina.

Gap junctions are dynamically modulated bridges allowing the transcellular passage of ions and small molecules with a molecular mass of up to 1 kDa, a mechanism utilized for molecular communication purposes by living cells. This same mechanism is also exploited by scientists to reveal the existence of gap junction contacts by the cell-to-cell movement of tracers. However, multiple labeling experiments require the availability of multiple gap junction-permeable tracers. To this end, we utilized the well-known transient OFF alpha retinal ganglion cell (RGC)-coupled array as a model system to study and compare the transjunctional movement of neurobiotin (NB), a commonly used tracer, and serotonin, a recently identified tracer. Although the transjunctional movement of serotonin has been established in cell cultures, here we show, for the first time, that serotonin is also a potent tracer in in vitro tissue. In addition, serotonin is lighter than the classical gap junction-permeable NB, and thus, we expected that tracer movement would be comparable to or better than that of serotonin. We found that intracellular serotonin injections result in the labeling of the coupled transient OFF alpha RGC array very similar to those of the classical NB-labeled arrays. Both serotonin and NB-injected transient OFF alpha RGCs displayed the well-known pattern with coupled RGCs and a cohort of coupled wide-field amacrine cells (ACs). By using morphological characteristics, we confirm that the serotonin and the NB-coupled AC arrays are identical, and thereby confirm that serotonin is a potent gap junction-permeable tracer and can be readily used as an alternative to NB in in vitro tissue. Moreover, serotonin can be utilized in parallel with other dyes or tracers, enabling the use of multiple labels in the same material.

Synaptoporin and parathyroid hormone 2 as markers of multimodal inputs to the auditory brainstem

The distribution of the synaptic vesicle protein synaptoporin was investigated by immunofluorescence in the central auditory system of the mouse brainstem. Synaptoporin immunostaining displayed region-specific differences. High and moderate accumulations of were seen in the superficial layer of the dorsal cochlear nucleus, dorsal and external regions of the inferior colliculus, the medial and dorsal divisions of the medial geniculate body and in periolivary regions of the superior olivary complex (SOC). Low or absent labeling was observed in the more central parts of these structures such as the principal nuclei of the SOC. It was conspicuous that dense synaptoporin immunoreactivity was detected predominantly in areas, which are known to be synaptic fields of multimodal, extra-auditory inputs. Target neurons of synaptoporin-positive synapses in the SOC were then identified by double-labelling immunofluorescence microscopy. We thereby detected synaptoporin puncta perisomatically at nitrergic, glutamatergic and serotonergic neurons but none next to neurons immunoreactive for choline-acetyltransferase and calcitonin-gene related peptide. These results leave open whether functionally distinct neuronal groups are accessed in the SOC by synaptoporin-containing neurons. The last part of our study sought to find out whether synaptoporin-positive neurons originate in the medial paralemniscal nucleus (MPL), which is characterized by expression of the peptide parathyroid hormone 2 (PTH2). Anterograde neuronal tracing upon injection into the MPL in combination with synaptoporin- and PTH2-immunodetection showed that (1) the MPL projects to the periolivary SOC using PTH2 as transmitter, (2) synaptoporin-positive neurons do not originate in the MPL, and (3) the close juxtaposition of synaptoporin-staining with either the anterograde tracer or PTH2 reflect concerted action of the different inputs to the SOC.

Construction and characterization of a synthesized herpes simplex virus H129-Syn-G2

Herpes simplex virus type 1 (HSV-1) causes lifelong infections worldwide, and currently there is no efficient cure or vaccine. HSV-1-derived tools, such as neuronal circuit tracers and oncolytic viruses, have been used extensively; however, further genetic engineering of HSV-1 is hindered by its complex genome structure. In the present study, we designed and constructed a synthetic platform for HSV-1 based on H129-G4. The complete genome was constructed from 10 fragments through 3 rounds of synthesis using transformation-associated recombination (TAR) in yeast, and was named H129-Syn-G2. The H129-Syn-G2 genome contained two copies of the gfp gene and was transfected into cells to rescue the virus. According to growth curve assay and electron microscopy results, the synthetic viruses exhibited more optimized growth properties and similar morphogenesis compared to the parental virus. This synthetic platform will facilitate further manipulation of the HSV-1 genome for the development of neuronal circuit tracers, oncolytic viruses, and vaccines.

Neural pathways from hypothalamic orexin neurons to the ventrolateral preoptic area mediate sleep impairments induced by conditioned fear.

Fear and sleep impairments common co-exist, but the underlying mechanisms remain unclear. Hypothalamic orexinergic neurons are involved in the regulation of sleep-wake and fear expression. The ventrolateral preoptic area (VLPO) is an essential brain region to promote sleep, and orexinergic axonal fibers projecting to the VLPO are involved in the maintenance of sleep-wake. Neural pathways from hypothalamic orexin neurons to the VLPO might mediate sleep impairments induced by conditioned fear. To verify above hypothesis, electroencephalogram (EEG) and electromyogram (EMG) were recorded for analysis of sleep-wake states before and 24 h after conditioned fear training. The retrograde tracing technique and immunofluorescence staining was used to identify the projections from the hypothalamic orexin neurons to the VLPO and to observe their activation in mice with conditioned fear. Moreover, optogenetic activation or inhibition of hypothalamic orexin-VLPO pathways was performed to observe whether the sleep-wake can be regulated in mice with conditioned fear. Finally, orexin-A and orexin receptor antagonist was administered into the VLPO to certify the function of hypothalamic orexin-VLPO pathways on mediating sleep impairments induced by conditioned fear. It was found that there was a significant decrease in the non-rapid eye movement (NREM) and rapid eye movement (REM) sleep time and a significant increase in the wakefulness time in mice with conditioned fear. The results of retrograde tracing technique and immunofluorescence staining showed that hypothalamic orexin neurons projected to the VLPO and observed the CTB labeled orexin neurons were significantly activated (c-Fos+) in the hypothalamus in mice with conditioned fear. Optogenetic activation of hypothalamic orexin to the VLPO neural pathways significantly decreased NREM and REM sleep time and increased wakefulness time in mice with conditioned fear. A significant decrease in NREM and REM sleep time and an increase in wakefulness time were observed after the injection of orexin-A into the VLPO, and the effects of orexin-A in the VLPO were blocked by a pre-administrated dual orexin antagonist (DORA). These findings suggest that the neural pathways from hypothalamic orexinergic neurons to the VLPO mediate sleep impairments induced by conditioned fear.