Glia Cell Morphology Analysis Using the Fiji GliaMorph Toolkit

Glial cells are the support cells of the nervous system. Glial cells typically have elaborate morphologies that facilitate close contacts with neighboring neurons, synapses, and the vasculature. In the retina, Müller glia (MG) are the principal glial cell type that supports neuronal function by providing a myriad of supportive functions via intricate cell morphologies and precise contacts. Thus, complex glial morphology is critical for glial function, but remains challenging to resolve at a sub-cellular level or reproducibly quantify in complex tissues. To address this issue, we developed GliaMorph as a Fiji-based macro toolkit that allows 3D glial cell morphology analysis in the developing and mature retina. As GliaMorph is implemented in a modular fashion, here we present guides to (a) setup of GliaMorph, (b) data understanding in 3D, including z-axis intensity decay and signal-to-noise ratio, (c) pre-processing data to enhance image quality, (d) performing and examining image segmentation, and (e) 3D quantification of MG features, including apicobasal texture analysis. To allow easier application, GliaMorph tools are supported with graphical user interfaces where appropriate, and example data are publicly available to facilitate adoption. Further, GliaMorph can be modified to meet users’ morphological analysis needs for other glial or neuronal shapes. Finally, this article provides users with an in-depth understanding of data requirements and the workflow of GliaMorph. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Download and installation of GliaMorph components including example data Basic Protocol 2: Understanding data properties and quality 3D—essential for subsequent analysis and capturing data property issues early Basic Protocol 3: Pre-processing AiryScan microscopy data for analysis Alternate Protocol: Pre-processing confocal microscopy data for analysis Basic Protocol 4: Segmentation of glial cells Basic Protocol 5: 3D quantification of glial cell morpholog

The Mast Cell Degranulator Compound 48/80 Directly Activates Neurons

Background Compound 48/80 is widely used in animal and tissue models as a “selective” mast cell activator. With this study we demonstrate that compound 48/80 also directly activates enteric neurons and visceral afferents. Methodology/Principal Findings We used in vivo recordings from extrinsic intestinal afferents together with Ca++ imaging from primary cultures of DRG and nodose neurons. Enteric neuronal activation was examined by Ca++ and voltage sensitive dye imaging in isolated gut preparations and primary cultures of enteric neurons. Intraluminal application of compound 48/80 evoked marked afferent firing which desensitized on subsequent administration. In egg albumen-sensitized animals, intraluminal antigen evoked a similar pattern of afferent activation which also desensitized on subsequent exposure to antigen. In cross-desensitization experiments prior administration of compound 48/80 failed to influence the mast cell mediated response. Application of 1 and 10 µg/ml compound 48/80 evoked spike discharge and Ca++ transients in enteric neurons. The same nerve activating effect was observed in primary cultures of DRG and nodose ganglion cells. Enteric neuron cultures were devoid of mast cells confirmed by negative staining for c-kit or toluidine blue. In addition, in cultured enteric neurons the excitatory action of compound 48/80 was preserved in the presence of histamine H1 and H2 antagonists. The mast cell stabilizer cromolyn attenuated compound 48/80 and nicotine evoked Ca++ transients in mast cell-free enteric neuron cultures. Conclusions/Significance The results showed direct excitatory action of compound 48/80 on enteric neurons and visceral afferents. Therefore, functional changes measured in tissue or animal models may involve a mast cell independent effect of compound 48/80 and cromolyn

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Mechanical stress activates neurites and somata of myenteric neurons

The particular location of myenteric neurons, sandwiched between the 2 muscle layers of the gut, implies that their somata and neurites undergo mechanical stress during gastrointestinal motility. Existence of mechanosensitive enteric neurons (MEN) is undoubted but many of their basic features remain to be studied. In this study, we used ultra-fast neuroimaging to record activity of primary cultured myenteric neurons of guinea pig and human intestine after von Frey hair evoked deformation of neurites and somata. Independent component analysis was applied to reconstruct neuronal morphology and follow neuronal signals. Of the cultured neurons 45% (114 out of 256, 30 guinea pigs) responded to neurite probing with a burst spike frequency of 13.4 Hz. Action potentials generated at the stimulation site invaded the soma and other neurites. Mechanosensitive sites were expressed across large areas of neurites. Many mechanosensitive neurites appeared to have afferent and efferent functions as those that responded to deformation also conducted spikes coming from the soma. Mechanosensitive neurites were also activated by nicotine application. This supported the concept of multifunctional MEN. 14% of the neurons (13 out of 96, 18 guinea pigs) responded to soma deformation with burst spike discharge of 17.9 Hz. Firing of MEN adapted rapidly (RAMEN), slowly (SAMEN) or ultra-slowly (USAMEN). The majority of MEN showed SAMEN behavior although significantly more RAMEN occurred after neurite probing. Cultured myenteric neurons from human intestine had similar properties. Compared to MEN, dorsal root ganglion neurons were activated by neurite but not by soma deformation with slow adaptation of firing. We demonstrated that MEN exhibit specific features very likely reflecting adaptation to their specialized functions in the gut

Sensitivity to Strain and Shear Stress of Isolated Mechanosensitive Enteric Neurons

Within the enteric nervous system, the neurons in charge to control motility of the gastrointestinal tract reside in a particular location nestled between two perpendicular muscle layers which contract and relax. We used primary cultured myenteric neurons of male guinea pigs to study mechanosensitivity of enteric neurons in isolation. Ultrafast Neuroimaging with a voltage-sensitive dye technique was used to record neuronal activity in response to shear stress and strain. Strain was induced by locally deforming the elastic cell culture substrate next to a neuron. Measurements showed that substrate strain was mostly elongating cells. Shear stress was exerted by hydrodynamic forces in a microchannel. Both stimuli induced excitatory responses. Strain activated 14% of the stimulated myenteric neurons that responded with a spike frequency of 1.9 (0.7/3.2) Hz, whereas shear stress excited only a few neurons (5.6%) with a very low spike frequency of 0 (0/0.6) Hz. Thus, shear stress does not seem to be an adequate stimulus for mechanosensitive enteric neurons (MEN) while strain activates enteric neurons in a relevant manner. Analyzing the adaptation behavior of MEN showed that shear stress activated rapidly/slowly/ultraslowly adapting MEN (2/62/36%) whereas strain only slowly (46%) and ultraslowly (54%) MEN. Paired experiments with strain and normal stress revealed three mechanosensitive enteric neuronal populations: one strain-sensitive (37%), one normal stress-sensitive (17%) and one strain- and stress-sensitive (46%).These results indicate that shear stress does not play a role in the neuronal control of motility but normal stress and strain

Compound 48/80 (c48/80) evoked Ca⁺⁺ transients in primary cultured enteric neurons. A

<p>shows two images of a Fluo-4 AM stained ganglion before c48/80 application and at the time of the maximal response. <b>B</b> illustrates the Ca⁺⁺ signal of the neuron marked by an arrow in A. Two consecutive c48/80 applications (marked by the bars below the traces) evoked comparable responses. In this ganglion all 8 neurons responded to c48/80. <b>C</b> confirms that responses to c48/80 application were not caused by activation of mechanosensors because spritz application of Krebs solution using the same application parameters had no effect. <b>D</b> illustrates that c48/80 evoked [Ca⁺⁺]_i increase is concentration dependent.</p

Compound 48/80 (c48/80) evoked activation of visceral afferents <i>in vivo</i>. A

<p>Jejunal afferent discharge stimulated by c48/80 administered intraluminally. A second administration 5 min later evoked a similar afferent response but subsequent administrations are completely desensitized, despite similar mechanosensitivity during the luminal perfusions. <b>B</b> In sensitized animals intraluminal application of the antigen egg albumin (EA) evoked a response similar to that observed with c48/80 and which too rapidly desensitized so that subsequent antigen failed to evoke a response. <b>C</b> Following desensitization to c48/80, intraluminal antigen still evoked a response similar to that in naïve control animals. When the order of administration was reversed and the response to antigen was desensitized, c48/80 was still able to evoke a response <b>D</b> illustrates mean data from these cross-desensitization experiments. The left panel shows the afferent response to saline or antigen in sensitized animals that had received pretreatment with vehicle (saline, N = 11) or c48/80 (N = 5). The panel on the right shows similar data for the response to vehicle (saline) or c48/80 in naïve control animals (N = 9) or sensitized animals following desensitization to antigen (N = 6). The response to c48/80 was significantly augmented after desensitization to antigen (P<0.01).</p