Labeling Of Neurons Research Articles

Resolution of Cerebral Inflammation Following Subarachnoid Hemorrhage

BackgroundAneurismal subarachnoid hemorrhage (SAH) is a type of hemorrhagic stroke that, despite improvement through therapeutic interventions, remains a devastating cerebrovascular disorder that has a high mortality rate and causes long-term disability. Cerebral inflammation after SAH is promoted through microglial accumulation and phagocytosis. Furthermore, proinflammatory cytokine release and neuronal cell death play key roles in the development of brain injury. The termination of these inflammation processes and restoration of tissue homeostasis are of utmost importance regarding the possible chronicity of cerebral inflammation and the improvement of the clinical outcome for affected patients post SAH. Thus, we evaluated the inflammatory resolution phase post SAH and considered indications for potential tertiary brain damage in cases of incomplete resolution.MethodsSubarachnoid hemorrhage was induced through endovascular filament perforation in mice. Animals were killed 1, 7 and 14 days and 1, 2 and 3 months after SAH. Brain cryosections were immunolabeled for ionized calcium-binding adaptor molecule-1 to detect microglia/macrophages. Neuronal nuclei and terminal deoxyuridine triphosphate-nick end labeling staining was used to visualize secondary cell death of neurons. The gene expression of various proinflammatory mediators in brain samples was analyzed by quantitative polymerase chain reaction.ResultsWe observed restored tissue homeostasis due to decreased microglial/macrophage accumulation and neuronal cell death 1 month after insult. However, the messenger RNA expression levels of interleukin 6 and tumor necrosis factor α were still elevated at 1 and 2 months post SAH, respectively. The gene expression of interleukin 1β reached its maximum on day 1, whereas at later time points, no significant differences between the groups were detected.ConclusionsBy the herein presented molecular and histological data we provide an important indication for an incomplete resolution of inflammation within the brain parenchyma after SAH. Inflammatory resolution and the return to tissue homeostasis represent an important contribution to the disease’s pathology influencing the impact on brain damage and outcome after SAH. Therefore, we consider a novel complementary or even superior therapeutic approach that should be carefully rethought in the management of cerebral inflammation after SAH. An acceleration of the resolution phase at the cellular and molecular levels could be a potential aim in this context.

Direct fluorescent labeling of NF186 and NaV1.6 in living primary neurons using bioorthogonal click chemistry.

The axon initial segment (AIS) is a highly specialized neuronal compartment that regulates the generation of action potentials and maintenance of neuronal polarity. Live imaging of the AIS is challenging due to the limited number of suitable labeling methods. To overcome this limitation, we established a novel approach for live labeling of the AIS using unnatural amino acids (UAAs) and click chemistry. The small size of UAAs and the possibility of introducing them virtually anywhere into target proteins make this method particularly suitable for labeling of complex and spatially restricted proteins. Using this approach, we labeled two large AIS components, the 186 kDa isoform of neurofascin (NF186; encoded by Nfasc) and the 260 kDa voltage-gated Na+ channel (NaV1.6, encoded by Scn8a) in primary neurons and performed conventional and super-resolution microscopy. We also studied the localization of epilepsy-causing NaV1.6 variants with a loss-of-function effect. Finally, to improve the efficiency of UAA incorporation, we developed adeno-associated viral (AAV) vectors for click labeling in neurons, an achievement that could be transferred to more complex systems such as organotypic slice cultures, organoids, and animal models.

Labeling PIEZO2 activity in the peripheral nervous system

Sensory neurons detect mechanical forces from both the environment and internal organs to regulate physiology. PIEZO2 is a mechanosensory ion channel critical for touch, proprioception, and bladder stretch sensation, yet its broad expression in sensory neurons suggests it has undiscovered physiological roles. To fully understand mechanosensory physiology, we must know where and when PIEZO2-expressing neurons detect force. The fluorescent styryl dye FM 1-43 was previously shown to label sensory neurons. Surprisingly, we find that the vast majority of FM 1-43 somatosensory neuron labeling in mice invivo is dependent on PIEZO2 activity within the peripheral nerve endings. We illustrate the potential of FM 1-43 by using it to identify novel PIEZO2-expressing urethral neurons that are engaged by urination. These data reveal that FM 1-43 is a functional probe for mechanosensitivity via PIEZO2 activation invivo and will facilitate the characterization of known and novel mechanosensory processes in multiple organ systems.

Emotional- and cognitive-like responses induced by social defeat stress in male mice are modulated by the BNST, amygdala, and hippocampus.

Chronic exposure to social defeat stress (SDS) has been used to investigate the neurobiology of depressive- and anxiety-like responses and mnemonic processes. We hypothesized that these affective, emotional, and cognitive consequences induced by SDS are regulated via glutamatergic neurons located in the bed nucleus of the stria terminalis (BNST), amygdaloid complex, and hippocampus in mice. Here, we investigated the influence of chronic SDS on (i) the avoidance behavior assessed in the social interaction test, (ii) the anxiety-like behavior (e.g., elevated plus-maze, and open field tests) (iii) depressive-like behaviors (e.g., coat state, sucrose splash, nesting building, and novel object exploration tests), (iv) the short-term memory (object recognition test), (v) ΔFosB, CaMKII as well as ΔFosB + CaMKII labeling in neurons located in the BNST, amygdaloid complex, dorsal (dHPC) and the ventral (vHPC) hippocampus. The main results showed that the exposure of mice to SDS (a) increased defensive and anxiety-like behaviors and led to memory impairment without eliciting clear depressive-like or anhedonic effects; (b) increased ΔFosB + CaMKII labeling in BNST and amygdala, suggesting that both areas are strongly involved in the modulation of this type of stress; and produced opposite effects on neuronal activation in the vHPC and dHPC, i.e., increasing and decreasing, respectively, ΔFosB labeling. The effects of SDS on the hippocampus suggest that the vHPC is likely related to the increase of defensive- and anxiety-related behaviors, whereas the dHPC seems to modulate the memory impairment. Present findings add to a growing body of evidence indicating the involvement of glutamatergic neurotransmission in the circuits that modulate emotional and cognitive consequences induced by social defeat stress.

Metabolic Glycan Labeling in Primary Neurons Enabled by Unnatural Sugars with No S-Glyco-Modification.

It is of great interest to probe glycosylation in primary neuron cultures. However, per-O-acetylated clickable unnatural sugars, which have been routinely utilized in metabolic glycan labeling (MGL) for analyzing glycans, showed cytotoxicity to cultured primary neurons and thus led to the speculation that MGL was not compatible with primary neuron cell cultures. Here, we uncovered that neuron cytotoxicity of per-O-acetylated unnatural sugars was related to their reactions with protein cysteines via non-enzymatic S-glyco-modification. The modified proteins were enriched in biological functions related to microtubule cytoskeleton organization, positive regulation of axon extension, neuron projection development, and axonogenesis. We thus established MGL in cultured primary neurons without cytotoxicity using S-glyco-modification-free unnatural sugars including ManNAz, 1,3-Pr2ManNAz, and 1,6-Pr2ManNAz, which allowed for visualization of cell-surface sialylated glycans, probing the dynamics of sialylation, and large-scale identification of sialylated N-linked glycoproteins and the modification sites in primary neurons. Particularly, a total of 505 sialylated N-glycosylation sites distributed on 345 glycoproteins were identified by 1,6-Pr2ManNAz.

Neurogenic timing of the inferior olive subdivisions is related to the olivocerebellar projection topography

The olivocerebellar projection is organized into an intricate topographical connection from the inferior olive (IO) subdivisions to the longitudinally-striped compartments of cerebellar Purkinje Cells (PCs), to play an essential role in cerebellar coordination and learning. However, the central mechanisms for forming topography need to be clarified. IO neurons and PCs are generated during overlapping periods of a few days in embryonic development. Therefore, we examined whether their neurogenic timing is specifically involved in the olivocerebellar topographic projection relationship. First, we mapped neurogenic timing in the entire IO by using the neurogenic-tagging system of neurog2-CreER (G2A) mice and specific labeling of IO neurons with FoxP2. IO subdivisions were classified into three groups depending on their neurogenic timing range. Then, we examined the relationships in the neurogenic-timing gradient between IO neurons and PCs by labeling topographic olivocerebellar projection patterns and PC neurogenic timing. Early, intermediate, and late groups of IO subdivisions projected to late, intermediate, and early groups of the cortical compartments, respectively, except for a few particular areas. The results indicated that the olivocerebellar topographic relationship is essentially arranged according to the reverse neurogenic-timing gradients of the origin and target.

Rabies virus-based labeling of layer 6 corticothalamic neurons for two-photon imaging in vivo

Neocortical layer 6 (L6) is less understood than other more superficial layers, largely owing to limitations of performing high-resolution investigations invivo. Here, we show that labeling with the Challenge Virus Standard (CVS) rabies virus strain enables high-quality imaging of L6 neurons by conventional two-photon microscopes. CVS virus injection into the medial geniculate body can selectively label L6 neurons in the auditory cortex. Only three days after injection, dendrites and cell bodies of L6 neurons could be imaged across all cortical layers. Ca2+ imaging in awake mice showed that sound stimulation evokes neuronal responses from cell bodies with minimal contamination from neuropil signals. In addition, dendritic Ca2+ imaging revealed significant responses from spines and trunks across all layers. These results demonstrate a reliable method capable of rapid, high-quality labeling of L6 neurons that can be readily extended to other brain regions.

Comparison of the Nerve Regeneration Capacity and Characteristics between Sciatic Nerve Crush and Transection Injury Models in Rats

ObjectiveTo provide useful information for selecting the most appropriate peripheral nerve injury model for different research purposes in nerve injury and repair studies, and to compare nerve regeneration capacity and characteristics between them. MethodsSixty adult SD rats were randomly divided into two groups and underwent crush injury alone (group A, n = 30) or transection injury followed by surgical repair (group B, n = 30) of the right hind paw. Each group was subjected to the CatWalk test, gastrocnemius muscle evaluation, pain threshold measurement, electrophysiological examination, retrograde neuronal labeling, and quantification of nerve regeneration before and 7, 14, 21, and 28 days after injury. ResultsGait analysis showed that the recovery speed in group A was significantly faster than that in group B at 14 days. At 21 days, the compound muscle action potential of the gastrocnemius muscle in group A was significantly higher than that in group B, and the number of labeled motor neurons in group B was lower than that in group A. The number of new myelin sheaths and the g-ratio were higher in group A than in group B. There was a 7-day time difference in the regeneration rate between the two injury groups. ConclusionThe regeneration of nerve fibers was rapid after crush nerve injury, whereas the transection injury was relatively slow, which provides some ideas for the selection of clinical research models.

Cell-Surface Targeting of Fluorophores in Drosophila for Rapid Neuroanatomy Visualization.

Visualizing neuronal anatomy often requires labor-intensive immunohistochemistry on fixed and dissected brains. To facilitate rapid anatomical staining in live brains, we used genetically targeted membrane tethers that covalently link fluorescent dyes for in vivo neuronal labeling. We generated a series of extracellularly trafficked small-molecule tethering proteins, HaloTag-CD4 (Kirk et al. Front. Neurosci. 2021, 15, 754027) and SNAPf-CD4, which directly label transgene-expressing cells with commercially available ligand-substituted fluorescent dyes. We created stable transgenic Drosophila reporter lines, which express extracellular HaloTag-CD4 and SNAPf-CD4 with LexA and Gal4 drivers. Expressing these enzymes in live Drosophila brains, we labeled the expression patterns of various Gal4 driver lines recapitulating histological staining in live-brain tissues. Pan-neural expression of SNAPf-CD4 enabled the registration of live brains to an existing template for anatomical comparisons. We predict that these extracellular platforms will not only become a valuable complement to existing anatomical methods but will also prove useful for future genetic targeting of other small-molecule probes, drugs, and actuators.

Single-Cell Labeling Strategies to Dissect Neuronal Structures and Local Functions.

The brain network consists of ten billion neurons and is the most complex structure in the universe. Understanding the structure of complex brain networks and neuronal functions is one of the main goals of modern neuroscience. Since the seminal invention of Golgi staining, single-cell labeling methods have been among the most potent approaches for dissecting neuronal structures and neural circuits. Furthermore, the development of sparse single-cell transgenic methods has enabled single-cell gene knockout studies to examine the local functions of various genes in neural circuits and synapses. Here, we review non-transgenic single-cell labeling methods and recent advances in transgenic strategies for sparse single neuronal labeling. These methods and strategies will fundamentally contribute to the understanding of brain structure and function.

“Self-inactivating” rabies viruses are susceptible to loss of their intended attenuating modification

Monosynaptic tracing using rabies virus is an important technique in neuroscience, allowing brain-wide labeling of neurons directly presynaptic to a targeted neuronal population. A 2017 article reported the development of a noncytotoxic version-a major advance-based on attenuating the rabies virus by the addition of a destabilization domain to the C terminus of a viral protein. However, this modification did not appear to hinder the ability of the virus to spread between neurons. We analyzed two viruses provided by the authors and show here that both were mutants that had lost the intended modification, explaining the paper's paradoxical results. We then made a virus that actually did have the intended modification in at least the majority of virions and found that it did not spread efficiently under the conditions described in the original paper, namely, without an exogenous protease being expressed in order to remove the destabilization domain. We found that it did spread when the protease was supplied, although this also appeared to result in the deaths of most source cells by 3 wk postinjection. We conclude that the new approach is not robust but that it could become a viable technique given further optimization and validation.

Taste arbor structural variability analyzed across taste regions.

Taste ganglion neurons are functionally and molecularly diverse, but until recently morphological diversity was completely unexplored. Specifically, taste arbors (the portion of the neuron within the taste bud) vary in structure, but the reason for this variability is unclear. Here, we analyzed structural variability in taste arbors to determine which factors determine their morphological diversity. To characterize arbor morphology and its relationship to taste bud cells capable of transducing taste stimuli (taste-transducing cell) number and type, we utilized sparse cell genetic labeling of taste ganglion neurons in combination with whole-mount immunohistochemistry. Reconstruction of 151 taste arbors revealed variation in arbor size, complexity, and symmetry. Overall, taste arbors exist on a continuum of complexity, cannot be categorized into discrete morphological groups, and do not have stereotyped endings. Arbor size/complexity was not related to the size of the taste bud in which it was located or the type of taste-transducing cell contacted (membranes within 180nm). Instead, arbors could be broadly categorized into three groups: large asymmetrical arbors contacting many taste-transducing cells, small symmetrical arbors contacting one or two taste-transducing cells, and unbranched arbors. Neurons with multiple arbors had arbors in more than one of these categories, indicating that this variability is not an intrinsic feature of neuron type. Instead, we speculate that arbor structure is determined primarily by nerve fiber remodeling in response to cell turnover and that large asymmetrical arbors represent a particularly plastic state.

Cholinergic Boutons are Distributed Along the Dendrites and Somata of VIP Neurons in the Inferior Colliculus.

Cholinergic signaling shapes sound processing and plasticity in the inferior colliculus (IC), the midbrain hub of the central auditory system, but how cholinergic terminals contact and influence individual neuron types in the IC remains largely unknown. Using pharmacology and electrophysiology, we recently found that acetylcholine strongly excites VIP neurons, a class of glutamatergic principal neurons in the IC, by activating α3β4* nicotinic acetylcholine receptors (nAChRs). Here, we confirm and extend these results using tissue from mice of both sexes. First, we show that mRNA encoding α3 and β4 nAChR subunits is expressed in many neurons throughout the IC, including most VIP neurons, suggesting that these subunits, which are rare in the brain, are important mediators of cholinergic signaling in the IC. Next, by combining fluorescent labeling of VIP neurons and immunofluorescence against the vesicular acetylcholine transporter (VAChT), we show that individual VIP neurons in the central nucleus of the IC (ICc) are contacted by a large number of cholinergic boutons. Cholinergic boutons were distributed adjacent to the somata and along the full length of the dendritic arbors of VIP neurons, positioning cholinergic signaling to affect synaptic computations arising throughout the somatodendritic compartments of VIP neurons. In addition, cholinergic boutons were occasionally observed in close apposition to dendritic spines on VIP neurons, raising the possibility that cholinergic signaling also modulates presynaptic release onto VIP neurons. Together, these results strengthen the evidence that cholinergic signaling exerts widespread influence on auditory computations performed by VIP neurons and other neurons in the IC.

Monosynaptic restriction of the anterograde herpes simplex virus strain H129 for neural circuit tracing.

Identification of synaptic partners is a fundamental task for systems neuroscience. To date, few reliable techniques exist for whole brain labeling of downstream synaptic partners in a cell-type-dependent and monosynaptic manner. Herein, we describe a novel monosynaptic anterograde tracing system based on the deletion of the gene UL6 from the genome of a cre-dependent version of the anterograde Herpes Simplex Virus 1 strain H129. Given that this knockout blocks viral genome packaging and thus viral spread, we reasoned that co-infection of a HSV H129 ΔUL6 virus with a recombinant adeno-associated virus expressing UL6 in a cre-dependent manner would result in monosynaptic spread from target cre-expressing neuronal populations. Application of this system to five nonreciprocal neural circuits resulted in labeling of neurons in expected projection areas. While some caveats may preclude certain applications, this system provides a reliable method to label postsynaptic partners in a brain-wide fashion.

Activity-Dependent Labeling of Olfactory Sensory Neurons Using RNA Fluorescence In Situ Hybridization Followed by Phospho-S6 Immunofluorescence.

This microscope-based method allows demonstrating that an odorant receptor responded to an odorant in vivo. In sections of olfactory epithelium from odorant-exposed mice, the subpopulation of olfactory sensory neurons expressing a particular odorant receptor type is labeled using RNA fluorescence in situ hybridization. Sequential immunofluorescence against the phosphorylated S6 ribosomal subunit reveals the activated olfactory sensory neurons. The presence of double-labeled cells confirms that the particular odorant receptor type was activated by the odorant stimulation.

Role of peptidergic neurons in modulating acupoint sensitization caused by neck acute inflammatory pain in rats 肽能神经元对颈部急性炎性痛模型大鼠穴位敏化的调节作用

ObjectivePain is a health problem frequently reported in the clinical and general populations. Acupoint therapy is one of the most effective ways to treat pain; however, its mechanism remains unclear. Therefore, this study aimed to explore the relationship between distal acupoints, dorsal root ganglion (DRG) neurons, and spinal cord dorsal horn (SDH) neurons in neck acute inflammatory pain (NAIP). MethodsNAIP model rats were used to explore the relationship between acupoint sensitization and pain. Out of fourteen rats, ten rats were grouped into control and NAIP groups, five rats in control and five rats in NAIP. Mustard oil was subcutaneously injected on one side between the C4 and C7 vertebrae of the neck to establish an NAIP model. Evans blue (EB) was injected through the tail vein to detect sensitized acupoints after NAIP modeling. EB exudation in the body, Lieque (LU7), and Lingdao (HT4) were evaluated. An immunofluorescence assay was conducted to detect the expression of calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), and c-Fos in the dorsal root ganglion (DRG) and spinal cord dorsal medullae spinalis (CDMS). Four rats were used for the retrograde labeling of neurons of the LU7 region to the DRG and CDMS using CTB-488 and CTB-555 microinjection. ResultsNAIP was shown to lead to oozing and pain sensitization in the LU7 and HT4 and increased the proportion of c-Fos+/CGRP+ and c-Fos+/IB4+ cells in both the DRG and CDMS. CTB-488 and CTB-555 injected into the LU7 and sensitization point areas were observed in the DRG and CDMS regions of NAIP rats. ConclusionsOur study revealed that NAIP could lead to oozing and pain sensitization in the LU7 and HT4 regions and that the pain point around LU7 might result from the transfer of peptidergic (CGRP-positive) and non-peptidergic (IB4-positive) neurons in the DRG and CDMS.

Anatomical analysis of sensory ganglia in mice

Ganglia are nodular structures in the peripheral nervous system (PNS) that are formed by a cluster of neuron somas. They have important roles in communicating signals from peripheral organs to the nervous system and work as a unique structure of the PNS. As great advances in neuronal manipulation and recording techniques have been made, an increasing amount of research is focused on the function of ganglia. However, ganglion surgery is still very challenging, particularly in small animals such as mice. In the present study, we performed anatomical analyses of two types of sensory ganglia, the dorsal root ganglia and the trigeminal ganglia. Hematoxylin and eosin (H&E) staining revealed structural differences between ganglia and nerve fibers. Immunofluorescence staining with antibodies to neuronal nuclear antigen (NeuN), a neuron-specific marker, labels neurons in ganglia and distinguishes ganglia from other tissues, such as lymph nodes. This study describes the anatomical analysis of the dorsal root ganglia and the trigeminal ganglia, which provides an anatomical basis for functional studies.

Magnetic Systems for Regenerative Medicine

Magnetic Systems for Regenerative Medicine

AAV13 Enables Precise Targeting of Local Neural Populations.

As powerful tools for local gene delivery, adeno-associated viruses (AAVs) are widely used for neural circuit studies and therapeutical purposes. However, most of them have the characteristics of large diffusion range and retrograde labeling, which may result in off-target transduction during in vivo application. Here, in order to achieve precise gene delivery, we screened AAV serotypes that have not been commonly used as gene vectors and found that AAV13 can precisely transduce local neurons in the brain, with a smaller diffusion range than AAV2 and rigorous anterograde labeling. Then, AAV13-based single-viral and dual-viral strategies for sparse labeling of local neurons in the brains of C57BL/6 or Cre transgenic mice were developed. Additionally, through the neurobehavioral test in the ventral tegmental area, we demonstrated that AAV13 was validated for functional monitoring by means of carrying Cre recombinase to drive the expression of Cre-dependent calcium-sensitive indicator. In summary, our study provides AAV13-based toolkits for precise local gene delivery, which can be used for in situ small nuclei targeting, sparse labeling and functional monitoring.

Motor learning drives dynamic patterns of intermittent myelination on learning-activated axons.

Myelin plasticity occurs when newly formed and pre-existing oligodendrocytes remodel existing patterns of myelination. Myelin remodeling occurs in response to changes in neuronal activity and is required for learning and memory. However, the link between behavior-induced neuronal activity and circuit-specific changes in myelination remains unclear. Using longitudinal in vivo two-photon imaging and targeted labeling of learning-activated neurons in mice, we explore how the pattern of intermittent myelination is altered on individual cortical axons during learning of a dexterous reach task. We show that behavior-induced myelin plasticity is targeted to learning-activated axons and occurs in a staged response across cortical layers in the mouse primary motor cortex. During learning, myelin sheaths retract, which results in lengthening of nodes of Ranvier. Following motor learning, addition of newly formed myelin sheaths increases the number of continuous stretches of myelination. Computational modeling suggests that motor learning-induced myelin plasticity initially slows and subsequently increases axonal conduction speed. Finally, we show that both the magnitude and timing of nodal and myelin dynamics correlate with improvement of behavioral performance during motor learning. Thus, learning-induced and circuit-specific myelination changes may contribute to information encoding in neural circuits during motor learning.