We investigated the synaptic innervation of apical dendrites of cortical pyramidal cells in a region between layers (L) 1 and 2 using 3-D electron microscopy applied to four cortical regions in mouse. We found the relative inhibitory input at the apical dendrite’s main bifurcation to be more than 2-fold larger for L2 than L3 and L5 thick-tufted pyramidal cells. Towards the distal tuft dendrites in upper L1, the relative inhibitory input was at least about 2-fold larger for L5 pyramidal cells than for all others. Only L3 pyramidal cells showed homogeneous inhibitory input fraction. The inhibitory-to-excitatory synaptic ratio is thus specific for the types of pyramidal cells. Inhibitory axons preferentially innervated either L2 or L3/5 apical dendrites, but not both. These findings describe connectomic principles for the control of pyramidal cells at their apical dendrites and support differential computational properties of L2, L3 and subtypes of L5 pyramidal cells in cortex. The apical dendrites of pyramidal neurons, the most abundant cell type in the mammalian cerebral cortex , have be3 and 66 × 89 × 202 µm3 , secondary visual (V2), posterior parietal (PPC) and anterior cingulate cortex (ACC) sized between 72 × 93 × 141 µm 202 µm3  at a voxpy (SBEM ). The imWe then identified all the synapses onto 82 ADs within 10 µm around their main bifurcation . Togethe−11).Next, we analyzed the synaptic input to L2 pyramidal cells’ ADs . Here, uFor this analysis, we identified putative inhibitory versus excitatory synapses by their location on dendritic shafts versus spines . To estWe next investigated whether the observed difference in putative inhibitory synapse density for L2 versus DL ADs could be counteracted by differences in synaptic size . SynapsA fraction of dendritic spines in cortex have been reported to be innervated by two or more synapses, with the secondary innervation often inhibitory . A high −4). L2 neurons, even though they had larger diameters, still received 59% higher absolute putative inhibitory synapse density per unit surface area compared to their DL counterparts . Conversely, axons seeded at L2 ADs did not innervate their DL counterparts to a substantial fraction .Next, we investigated to what degree the innervation of individual apical dendrites by multiple synapses from the same inhibitory axon contributed to the establishment of axonal target preference for types of ADs . Axons tSince we had acquired four 3D EM datasets from different parts of the cerebral cortex including primary sensory (S1), secondary sensory (V2), and higher-order  areas , we thenWe next wanted to determine whether the observed innervation differences were common to supragranular (L2/3) vs infragranular (L5) pyramidal cells , or whether the putative inhibitory innervation was in fact distinct between L2 and the other pyramidal cells . We usedIn order to also differentiate between L5 pyramidal cells termed ‘thick tufted’ and ‘slender tufted’ , we used−4, Post-hoc Tukey’s range test, p = 2.7 × 10−5 and 0.002 for L2 vs. L3 and L5tt, respectively).When we then analyzed the fractional putative inhibitory synapse densities at the main bifurcation for L2, L3, L5tt and L5st pyramidal cells , we foun2 = 0.74, p < 10−5). Using only the linear fit to excitatory synapse density and the average putative inhibitory synapse density, 55% variability in the putative inhibitory fraction of L5 neurons could be explained .The putative inhibitory synapse fraction in L5 pyramidal cells was strongly anti-correlated to the ‘thick-tuftedness’ of the L5 pyramidal cell . This dependence was even evident within the population of L2 pyramidal cells, consistent with a possible synaptic specificity mechanism that is dependent on the distance to the cell body. Interestingly, this dependence was weaker for the absolute density of excitatory or putative inhibitory synapses, compared to their ratios . The relationship was consistent for all pyramidal cell types except L5st . For L5st pyramidal cells, the putative inhibitory input fraction at their distal apical tufts was also higher than their main bifurcations, with about a third of their synapses putative inhibitory  while the excitatory input increases by ~1.5 fold, yielding an about 3-fold reduced putative inhibitory input fraction towards their distal tufts (−4).For L2 pyramidal cells, to the contrary, putative inhibitory synapse density decreases ~2 fold towards the distal tuft . Only LIn conclusion, our findings describe non-random connectivity at the apical tuft input domain of pyramidal neurons in cortex. They imply unique innervation patterns for the apical dendrites of L2 vs L3 vs L5 pyramidal cells yielding highly variable and cell-type specific putative inhibitory-to-excitatory synaptic input compositions, and suggest differential modes of inhibitory operation for the main classes of pyramidal cells in the cortex.All experimental procedures were performed according to the law of animal experimentation issued by the German Federal Government under the supervision of local ethics committees and according to the guidelines of the Max Planck Society. The experimental procedures were approved by Regierungspräsidium Darmstadt, under protocol ID V54 - 19c20/15 F126/1015  or V54 – 19 c 20/15 – F126/1002 . The S1 sample was prepared following experimental procedures approved by Regierung von Oberbayern, 55.2-1-54-2532.3-103-12.The cortical tissue processing, 3D electron microscopy and data alignment for S1 and LPtA samples were described previously in 2 (Sigma-Aldrich) in 80 mM cacodylate buffer (pH = 7.4). The animal was decapitated and the skull was removed to expose the brain. The head was next submerged in fixative solution overnight at 4°C.Adult (P56 – 57) wild type mice (C57BL/6J) were injected with general analgesia  and 100 mg/kg Metamizol ). Next, animals were anesthetized by inhalation of isoflurane (3–3.5% in carbogen) and were perfused transcardially using 15 ml of cacodylate buffer  followed by 30 ml of fixative solution at a flow rate of 10 ml/min. The fixative solution was 2.5% PFA , 1.25% glutaraldehyde (Serva) and 0.5% CaClFirst, a 600 µm coronal slice containing the region of interest was acquired using a vibrating microtome  guided by a reference atlas . Next, w4  in cacodylate buffer for 90 min. The sample was then treated with 2.5% ferrocyanide  in cacodylate buffer for 90 min and 2% buffered OsO4 for 45 min. We then rinsed the tissue in cacodylate and ultrapure water  for 30 min each. Osmium content of the sample was amplified by treatment with saturated aqueous thiocarbohydrazide  and 2% aqueous OsO4 for 60 and 90 min, respectively. The sample was moved to 2% uranyl acetate (Serva) solution for overnight incubation at 4°C. The following day, the tissue, still in uranyl acetate, was warmed to 50°C for 120 min in an oven . This was followed by incubation in lead aspartate at 50°C for 120 min. The lead aspartate solution was prepared by dissolving 0.066 g lead nitrate  in a 10 ml 0.03 M aspartic acid  solution and adjusting the pH to 5.0.Samples were prepared for serial block-face electron microscopy following a slightly modified en-bloc staining method as described in Next, the cortical tissue was dehydrated by incubation in 50–100% ethanol gradient . This was followed by at least three 20–45 min incubation steps in pure acetone . The sample was then transferred to a 1:1 mixture of acetone and Spurr’s resin  for 3–4 hr with slow/no rotation. Tubes were opened to allow for acetone evaporation overnight . At this stage, the PPC-2 sample was transferred to a 3:1 mixture of Spurr’s resin and acetone instead. The following day, the infiltration process continued for two 3 hr (PPC-2) or one 6 hr  incubation steps in pure Spurr’s resin mixture. The tissue was then transferred to a flat-embedding mold and cured at 70°C for at least 48 hr. Specific time and temperature of dehydration and embedding steps are detailed in Table 2, Note that all procedures were performed at room temperature in 2 ml reaction tubes  unless stated otherwise. In addition, the initial incubation steps (until dehydration) were performed with the aid of an automatic microwave tissue processor  for V2, PPC, and ACC samples. Finally, treatment steps were interleaved with two 30 min washing steps in ultrapure water (from TCH step until dehydration initiation).The samples were excised from the resin block and mounted on an aluminum pin using epoxy glue . They were then trimmed to a block-face area of ~750 µm x 750 µm using a diamond head trimming machine . In addition, tissue was sputter coated with 100–200 nm of gold  to increase conductivity and reduce charging artefacts.3 for PPC-2 and LPtA, respectively).The SBEM microtome (courtesy of W. Denk) was fit inside the door of a scanning electron microscope  routines. We used cross-correlation or speeded-up robust feature (SURF) detection in the overlap region to measure the relative shift between image patches. These patches were full image tiles  or tile sub regions . The position of each patch was then globally optimized using least-square regression. The image volume was then partitioned into 1024 × 1024 × 1024 voxel blocks and written into webKnossos  three-diThe PPC-2 dataset was aligned using the affine alignment method from The skeleton reconstruction and synapse annotations were saved as a NML (XML-based format) file. They were subsequently parsed into a MATLAB (release 2014b-2019a) class with node and edge list properties using custom-written C++ routines. Each node had accompanying attributes, such as, comment string and coordinate. These attributes were used to extract different features of the annotation as described in the following sections.Apical dendrites (ADs) were identified based on their radial direction and diameter (~1–3 µm). The soma morphology and axon initial segment direction (towards white matter (WM)) of candidate pyramidal cells were examined where possible.Within the S1, V2, PPC and ACC datasets, ADs were classified depending on the existence of soma in the image volume . ADs witThe depth of the apical dendrite’s source soma relative to pial surface was used to differentiate layer 2, 3 and 5 cells in the LPtA and PPC-2 datasets that were contained in layers 2–5 . To obtain an accurate estimate of soma depth relative to pial surface, the section thickness was corrected (by a factor of 1.49), assuming soma dimensions to be similar in-plane and along the cutting direction.The main bifurcation of apical dendrites was defined as the branching point with two daughter branches of similar thickness and branching angle , L2MN , L3 , L5tt , L5st  pyramidal cells and considered the path length of these annotations as the soma to main bifurcation distance .We binned the excitatory and putative inhibitory synapses based on their path distance to soma in the L2 pyramidal ADs (A random subset of axons targeting shaft or spine of ADs were reconstructed .We then classified axons into excitatory and putative inhibitory by using a 50% threshold on the single-innervated spine innervation fraction . SubsequA random subset of shaft and spine (n = 41 per AD type) synapses were used to determine the synaptic interface area  of the remaining axonal path within 20 µm tangential cortical slices  and deep layer  ADs. An expert annotator (JO or AK) then reconstructed the axons throughout the dataset. We also annotated all the other synapses and postsynaptic partners for each axon. In addition, reconstructions were checked by another expert annotator (JO or AK) for morphological irregularities such as sharp branching angles and untraced endings.The postsynaptic targets were categorized into one of the following: layer 2 or deep layer apical dendrite (shaft and spine of trunk and primary branches), shaft of other dendrites, single- or double-innervated (including neck targeting) spine, layer 2 cell body, axon initial segment (AIS) or glia. The innervation fraction of an axon was computed by dividing the number of synapses for each specific target by the total number of synapses of that axon (the seed synapse was excluded). The innervation fractions were averaged for each AD seed type . Each daTo measure the frequency of multiple innervation of an AD by inhibitory axons used in our investigation of axonal specificity, we annotated all the cases were such multi-innervation happened . We thenTo visualize the surface of the main bifurcation of PPC and S1 apical dendrites and axons, we segmented the image volume using SegEM  and collVolume of the AD shaft at main bifurcation in V2 and ACC datasets were generated by tracing the outline of the dendrites using the volume tracing mode in webKnossos and 3D data was processed in a similar fashion .Skeleton reconstructions of axonal  and dendThe EM ultra-structure of an AD, synapses and cell bodies was demonstrated by electron microscopy images (PPC and ACC datasets) of their cross-sections .The visualizations were generated using MATLAB (mathworks) and Amira .Significance of difference between L2 and DL AD excitatory and putative inhibitory synapse densities  and 2g, To identify significant differences in the axonal innervation fractions, a bootstrapping test was used. The specificities of the two axonal groups were concatenated and 10,000 bootstrap resamples were drawn to match the number of axons seeded from L2 and DL ADs. The mean difference in each bootstrap resample between L2 and DL groups was compared to the sample mean difference. The p-value was defined as the fraction of bootstrap resamples which had a value more extreme compared to the sample mean. The significance threshold was set to 0.05 with Bonferroni correction for eight comparisons .We used a multivariate analysis of variance (MANOVA) test to measure the effect of cortical region on inhibitory axonal innervation probability for six postsynaptic targets . Axon initial segment (AIS) and glia innervation fractions were excluded since they contained few synapses and resulted in singular matrices when MANOVA test was applied . The null hypothesis states that the differences between the mean fractional innervations of putative inhibitory axons in S1, V2, PPC and ACC is statistically insignificant. We followed the MANOVA test when the null hypothesis was rejected with multiple comparisons using Bonferroni corrected one-way ANOVA tests . We then2) was computed as a goodness of fit measure. We also used R2 to compare models explaining the relationship between thick-tuftedness  and the putative inhibitory fraction at the main bifurcation. To understand the relationship between morphology and synapse density of L5 neurons, we fitted a linear model to individual synapse densities and a linear fraction model  of the form  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study uses 3-D electron microscopy to map the inhibitory and excitatory synaptic input distribution of the apical dendrites of pyramidal cells in the mouse cortex. The authors compare excitatory/inhibitory synapse density in L2, L3 and L5 pyramidal cells in different cortical regions, at the bifurcation point from the apical dendritic trunk and the distal dendritic tufts. The resultant connectomic principles for the inputs onto pyramidal cells at their apical dendrites support specific computational properties of L2, L3 and subtypes of L5 pyramidal cells, and is a rich data set that should be of wide interest in the field.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Cell-type specific innervation of cortical pyramidal cells at their apical tufts" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The reviewers were in the main positive about your study mapping the inhibitory and excitatory synaptic input distribution of the apical dendrites of pyramidal cells in the mouse cerebral cortex. Your comparison of excitatory/inhibitory synapse density in L2, L3 and L5 pyramidal cells in different cortical regions, at the bifurcation point from the apical dendritic trunk and the distal dendritic tufts, provides a very rich data set, one that should be of great interest to the field.The reviewers found your results novel for the level of anatomical analysis and for implications of differential modes of inhibition for pyramidal cells in the cortex, including the following rules of synapse distribution along the apical dendrite: a. At the bifurcation point at the L1/2 border, inhibitory synapse density is higher in L2 cells than in deeper layer cells; b. At the distal apical tuft, the excitatory synapse density is lower in L5 cells than in L2 cells; c. Dendrites at the L1/2 border of L2 and deeper layer pyramidal cells are innervated by axons of different inhibitory cells. These results suggest that the computational properties of apical dendrites are distinct in L2 and L5 pyramidal cells.Essential revisions:Even with this praise, the reviewers had issues with the definition of the structures you analyzed, specifically, the criteria for classification of E and I synapses, for parsing orientation of distal dendrites, and for identifying axonal inputs. Many of these amendments can be textual, yet the first of these major points in particular, may affect your analysis.1) Classification of synapses: Two of the reviewers at first assumed that you had classified synapses by the classical definition, i.e. symmetric as inhibitory and asymmetric as excitatory, but you state that "Shaft synapses, synapses on spine necks and secondary spine innervations were treated as inhibitory. Primary spine innervations were treated as excitatory synapses ". a) Whereas one could conclude from the literature, that a single spine innervation represents an excitatory synapse, and a doubly innervated spine having one inhibitory and one excitatory input , the reasoning goes astray for shaft synapses, which have been shown to represent both, with recent studies indicating that 15-20% of asymmetric synapses (presumably glutamatergic) are made on the dendritic shaft in the upper layers of cortex.b) Figure 2E, F, G, indicates that some of axons innervating a shaft then also make a synapse on a spine without another input more frequently than on a spine with another input. Most spines with an inhibitory input usually have another excitatory input in the neocortex. The axons making a single synapse on spines may be thus be excitatory; please explain.c) Validation of your classification system is requested: Discuss the differences between your assumptions and the literature, and the possible impact of your assumptions on the presented connectomic analysis. Do you or others have evidence that shaft synapses are gephyrin-positive, for example, beyond the Lascone study you cite (in BioRxiv)?2) Identification of apical dendrites: a) You based this identification on radial direction and diameter. Whereas this may apply for deep dendrites, there might be some ambiguity for superficial cells. How exactly are L2 pyramids distinguished from other cell types in superficial layers? Can you provide quantitative characteristics of the dendrites analyzed, and possibly of some dendrites and their somata that were not considered to belong to pyramids? Such information would provide a baseline for future studies, and would avoid generalization and possible biases.b) You assumed that dendritic branches from thicker dendrites around the L1/2 border represent bifurcation points from an apical dendritic trunk. L2 pyramidal cells were reconstructed down to their somata, but deeper layer pyramidal cells not. L5 thick-tufted pyramidal cells frequently have the bifurcation points in the center of L2/3. Is it possible that only L5 thin-tufted cells are analyzed in this study. This sampling bias may affect the conclusions.c) The authors make no distinction between thick tufted L5 (possibly L5B) and slender layer 5 (possibly L5A). There are distinct functional differences between them. It would be interesting if the authors can distinguish them and quantify the proportion of inhibitory inputs onto these two types?3) Distinction between L2 and L3 pyramidal neurons:A classification of pyramidal cells based on the depth of the soma may not be reliable (e.g. L2 vs. L3). Both subtypes are possibly intermingled in the more superficial regions. Another concern might be that L2 cells without a main bifurcation are excluded according to the listed criteria, as well as branches of either type that run more obliquely or even parallel to the surface. Please comment on this, and provide more numeric data such as cell body position, size, possibly branch numbers, etc. for future reference, again, to avoid over-generalization of the difference between L2 and L3.4) Contact by axons: a) For the axons that were reconstructed, were multiple contacts with the same dendrite or perhaps even the same neuron excluded? Did this occur?b) When discussing the axon identity in the Results section, the authors report only the axons that targeted dendritic shafts or spines, and not axons that targeted neither. In Figure 1H and Supplementary Figure 1B, the question of whether all reported axons target contact at least one spine or shaft is important in order to better understand the results.5) Synapse density: a) You report excitatory and inhibitory densities as a function of distance, and do not report the density per unit area, which is more informative.b) The inhibitory synapse density around the border of L1/2 is different between L2 and L5 cells. The dendritic size around the border may be different between L2 and L5 cells. It would therefore be informative to show the relation between dendrite thickness and inhibitory synapse density of the dendrites at this border.eLife. Your revised article has been evaluated by Eve Marder as the Senior Editor, a Reviewing Editor and two peer reviewers.Thank you for resubmitting your work entitled "Cell-type specific innervation of cortical pyramidal cells at their apical dendrites" for further consideration by Both reviewers appreciated the revisions on your study mapping the inhibitory and excitatory synaptic input distribution of the apical dendrites of pyramidal cells in the mouse cortex. Your comparison of excitatory/inhibitory synapse density in L2, L3 and L5 pyramidal cells in different cortical regions, at the bifurcation point from the apical dendritic trunk and the distal dendritic tufts, is a rich data set that should be of wide interest in the field. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The reviewers still have a lingering concern: While they are receptive to the concept that if an axon maintains shaft synapses in more than 90% of its dendritic contacts, it likely represents an inhibitory axon, they and I suggest that you state this at the beginning of the Results section and that you did not use the classic symmetry in the pre- and postsynaptic densities to identify inhibitory synapses. The use of the term inhibitory synapses comes before you test this with seeding method, which makes the argument a bit circular. We suggest that you start by referring to shaft and spine synapses , and refer to inhibitory synapses only once you have made their verification argument (subsection “Shaft vs. spine synapses”). In the latter section you can then present a plot that contains the corrected densities or fractions. Alternatively, you could first develop the argument of Figure 2A, B, and then present the data that are currently in Figure 1, maintaining the term inhibitory – though we would prefer 'putative inhibitory' over 'inhibitory'.In the Discussion, you could also caution that identification of excitatory and inhibitory synapses using the classical asymmetrical or symmetrical criteria was difficult because the image quality of SBEM is slightly sub-par. New Figure 3 clearly indicates that some of axons innervating a shaft next make a synapse on a spine with (not without) another input more frequently than on a spine without another input. From the revised Figures 2-4, the readers can now easily understand the logic by which to differentiate the excitatory and inhibitory axons, not just based on the single postsynaptic structure, but on consideration of successive targets. Restating these points in the Discussion would strengthen your arguments.On a positive note, reviewer 2 wondered whether the apical dendrites analyzed here belong to thick or slender tufted pyramidal cells in layer 5 because the two types of pyramidal cells significantly differ in function. Your new analysis has revealed their differences in spine density and this reviewer applauded this finding, and also found your quantitative analysis and conclusion that dendritic size is a key factor in determining inhibitory synapse density a strong contribution. Essential revisions:[…] The reviewers had issues with the definition of the structures you analyzed, specifically, the criteria for classification of E and I synapses, for parsing orientation of distal dendrites, and for identifying axonal inputs. Many of these amendments can be textual, yet the first of these major points in particular, may affect your analysis.1) Classification of synapses: Two of the reviewers at first assumed that you had classified synapses by the classical definition, i.e. symmetric as inhibitory and asymmetric as excitatory, but you state that "Shaft synapses, synapses on spine necks and secondary spine innervations were treated as inhibitory. Primary spine innervations were treated as excitatory synapses ". a) Whereas one could conclude from the literature, that a single spine innervation represents an excitatory synapse, and a doubly innervated spine having one inhibitory and one excitatory input , the reasoning goes astray for shaft synapses, which have been shown to represent both, with recent studies indicating that 15-20% of asymmetric synapses (presumably glutamatergic) are made on the dendritic shaft in the upper layers of cortex.We fully agree with the point raised by the reviewers that a single shaft or spine synapse can in principle be excitatory or inhibitory. 3D EM data, however, allows us to reconstruct the axons presynaptic at these synapses; which provides the targets of all other of the output synapses of these axons. Therefore, by reconstructing the presynaptic axons, we can quantify in how many cases a shaft synapse is made by an axon that in fact prefers spine innervations, and vice versa. We used the reconstructions of 430 presynaptic axons from synapses of the apical dendrites in layers 1 and 2 , and determined their other synaptic targets. The distribution of the fraction of single-innervated spine targets of these axons were almost binary  with axons preferring either shaft or spines of dendrites with the exception of axons innervating the shafts of L5 slender tufted ADs . Quantitatively, 91 of 92 axons seeded from the shafts of L2 ADs made at least 80% of their other output synapses onto shafts, as well (and 89 of 92 axons made at least 90% of their other synapses onto shafts). Similarly, 34 of 35 axons seeded from spines at L2 ADs made at least 80% of their other synapses as primary spine innervations. Numbers for synapses seeded at DL ADs were comparable.We then calculated a corrected inhibitory fraction based on this connectivity criteria for classifying excitatory and inhibitory input and found it to be rather unchanged for most cell types . For L5 slender tufted ADs, synapse densities were corrected for the synapse identity  due to the substantial spine-preferring input onto their AD shaft (44-60%).In summary, while for single synapses, the identification of spine vs. shaft targets with the synaptic classification of excitatory vs. inhibitory is error prone, the classification of axonal type by preference of their other local output synapses yields misclassification rates of less than 7% ; it appears that the axons innervating ADs as studied here show a stronger bimodal preference for either shafts or spines than those reconstructed in L4, ).b) Figure 2E, F, G, indicates that some of axons innervating a shaft then also make a synapse on a spine without another input more frequently than on a spine with another input. Most spines with an inhibitory input usually have another excitatory input in the neocortex. The axons making a single synapse on spines may be thus be excitatory; please explain.This was an unfortunate error in former Figure 2. The labels have been corrected in current Figure 3D, 4B-C . The shaft-preferring axons (inhibitory) innervate mainly spines with an additional (presumably excitatory) synapse (91.35% of 821 spine synapses). They only innervate a spine without a partner 8.65% of the time. See also the additional analyses in Figure 2A-C described above.c) Validation of your classification system is requested: Discuss the differences between your assumptions and the literature, and the possible impact of your assumptions on the presented connectomic analysis. Do you or others have evidence that shaft synapses are gephyrin-positive, for example, beyond the Lascone study you cite (in BioRxiv)?Our connectivity metric for the identification of axonal input type suggests a very strong shaft- vs. spine- preference for axons innervating most pyramidal neurons . L5st neurons, however, did receive a significant amount of input from spine-preferring axons onto their shafts. We, therefore, corrected our synapse density measurement for this cell-type specific difference. We also reviewed a few studies which investigated the fraction of asymmetric and symmetric synapses  on shaft and spine of pyramidal neurons using 3D-EM:1)  used a combination of ssTEM with Teal-Gephyrin 2P imaging. The authors found 26/26 asymmetric synapses on spines (no shaft asymmetric innervation) and 6/10 inhibitory synapses on shaft of the L2/3 pyramidal neuron dendrite (length: 30 µm). 3/10 inhibitory synapses were located on double-innervated spines and 1/10 single-innervated spine.2)  studied the basal dendrites of the spiny stellate neurons in L4 of primary somatosensory cortex using serial section TEM (ssTEM). They found 100% of shaft and 3% (14/359) of spine synapses to be symmetric.3)  analyzed multiple segments of apical dendrites in L5/6 pyramidal neurons using ssTEM. They found 20% of shaft synapses to be asymmetric and only 0.6% of spine synapses to be symmetric ).In conclusion, the degree to which the spine/shaft preference proxies for identification of excitatory and inhibitory synapses is dendrite type specific and shall be studied in the specific region of study either using an estimation method as described here or dense reconstruction of axons where most axon could be identified based on their single-innervated spine innervation fraction.Notably, studies where dendrite types are not considered and a volume average of asymmetric and symmetric synapses is calculated could result in high degrees of symmetric input to shafts ).2) Identification of apical dendrites: a) You based this identification on radial direction and diameter. Whereas this may apply for deep dendrites, there might be some ambiguity for superficial cells. How exactly are L2 pyramids distinguished from other cell types in superficial layers? Can you provide quantitative characteristics of the dendrites analyzed, and possibly of some dendrites and their somata that were not considered to belong to pyramids? Such information would provide a baseline for future studies, and would avoid generalization and possible biases.To disambiguate the morphology of the apical dendrites used in our analysis, we reconstructed the shaft morphology of all pyramidal neurons used in our cell-type analysis . We also added diameter measurements . In addition, we specifically looked for pyramidal neurons with obliquely directed apical dendrites  and measured their inhibitory input and compared it to other L2 pyramidal neurons . In the two examples we found of L2 marginal pyramidal neurons, we did not find evidence that their input fraction was different from L2 pyramidal cells. For the distinction of L2 and L3 pyramidal cells, we again were able to use our larger EM datasets for clear differentiation .b) You assumed that dendritic branches from thicker dendrites around the L1/2 border represent bifurcation points from an apical dendritic trunk. L2 pyramidal cells were reconstructed down to their somata, but deeper layer pyramidal cells not. L5 thick-tufted pyramidal cells frequently have the bifurcation points in the center of L2/3. Is it possible that only L5 thin-tufted cells are analyzed in this study. This sampling bias may affect the conclusions.We reconstructed all the pyramidal neurons used in our cell-type analysis in the high- and low-resolution segments of LPtA and PPC-2 datasets down to their soma of origin for L2-5 neurons . We found the AD morphology of the former L5 group much closer to L5B  neurons. Next, we identified six new L5 neurons with smaller soma and slender dendrite morphology  . Next, we used hierarchical clustering to classify L5 neurons based on their soma diameter, AD trunk diameter, number of oblique dendrites and main bifurcation depth . In addition, these neurons had a substantially lower spine density . L5st neurons also seemed to have an increased inhibitory input fraction (15-31%) as compared to L5tt neurons in upper cortex .c) The authors make no distinction between thick tufted L5 (possibly L5B) and slender layer 5 (possibly L5A). There are distinct functional differences between them. It would be interesting if the authors can distinguish them and quantify the proportion of inhibitory inputs onto these two types?We thank the reviewers for the insightful suggestion – the additional analyses ofL5 slender tufted cells yielded an interesting specialization in input fraction .3) Distinction between L2 and L3 pyramidal neurons:A classification of pyramidal cells based on the depth of the soma may not be reliable (e.g. L2 vs. L3). Both subtypes are possibly intermingled in the more superficial regions. Another concern might be that L2 cells without a main bifurcation are excluded according to the listed criteria, as well as branches of either type that run more obliquely or even parallel to the surface. Please comment on this, and provide more numeric data such as cell body position, size, possibly branch numbers, etc. for future reference, again, to avoid over-generalization of the difference between L2 and L3.We explicitly looked for L2 pyramidal neurons with an apical dendrite running oblique to the pial surface and found two of these marginal neurons . We also compared their synaptic input composition with other L2 neurons . Our preliminary analysis (n=2) fails to rule out the possibility that L2MN neurons have a similar inhibitory fraction to other L2 neurons. For the distinction between L2 and L3, please see the new cell type gallery. Also, we report the relation between synaptic input composition and cell body depth, and AD length , which should avoid any overly binary distinctions.4) Contact by axons: a) For the axons that were reconstructed, were multiple contacts with the same dendrite or perhaps even the same neuron excluded? Did this occur?We analyzed the frequency of multiple contacts with ADs  and its effect on our conclusions. Multiple contacts within the EM volumes we analyzed occurred in 23% of the cases, such that the preference of axons for innervating certain types of ADs remained rather substantial even when disregarding the multiple innervations .b) When discussing the axon identity in the Results section, the authors report only the axons that targeted dendritic shafts or spines, and not axons that targeted neither. In Figure 1H and Supplementary Figure 1B, the question of whether all reported axons target contact at least one spine or shaft is important in order to better understand the results.All axons reported in the manuscript were seeded from either spine or shaft synapses of an AD. Thus, they innervated at least the shaft or spine of the seed dendrite. For their remaining targets, , we identified these for a subset of axons, and report the distribution of axonal targets in Figure 3D and Figure 4 – but always for axons that targeted at least one spine or one shaft of an AD. We added a statement to that end to the Results subsection “Comparative analysis of innervation preference between cortex types”.5) Synapse density: a) You report excitatory and inhibitory densities as a function of distance, and do not report the density per unit area, which is more informative.We thank the reviewers for this suggestion. We additionally measured the diameter of apical dendrites and used it to calculate the synapse density per unit surface area of each cell type . All conclusions remained unaltered when normalizing for surface area of apical dendrites instead of their shaft path length.b) The inhibitory synapse density around the border of L1/2 is different between L2 and L5 cells. The dendritic size around the border may be different between L2 and L5 cells. It would therefore be informative to show the relation between dendrite thickness and inhibitory synapse density of the dendrites at this border.We added the relationship between the apical dendrite diameter and inhibitory/excitatory synapse density for apical dendrites of different cell types . We found a correlation between inhibitory synapse density and the diameter of the apical dendrite. However, the diameter differences were not substantial enough to change the patterns of synapse density observed using the distance-based metric .[…] The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The reviewers still have a lingering concern: While they are receptive to the concept that if an axon maintains shaft synapses in more than 90% of its dendritic contacts, it likely represents an inhibitory axon, they and I suggest that you state this at the beginning of the Results section and that you did not use the classic symmetry in the pre- and postsynaptic densities to identify inhibitory synapses. The use of the term inhibitory synapses comes before you test this with seeding method, which makes the argument a bit circular. We suggest that you start by referring to shaft and spine synapses , and refer to inhibitory synapses only once you have made their verification argument (subsection “Shaft vs. spine synapses”). In the latter section you can then present a plot that contains the corrected densities or fractions. Alternatively, you could first develop the argument of Figure 2A, B, and then present the data that are currently in Figure 1, maintaining the term inhibitory – though we would prefer 'putative inhibitory' over 'inhibitory'.In the Discussion, you could also caution that identification of excitatory and inhibitory synapses using the classical asymmetrical or symmetrical criteria was difficult because the image quality of SBEM is slightly sub-par. New Figure 3 clearly indicates that some of axons innervating a shaft next make a synapse on a spine with (not without) another input more frequently than on a spine without another input. From the revised Figures 2-4, the readers can now easily understand the logic by which to differentiate the excitatory and inhibitory axons, not just based on the single postsynaptic structure, but on consideration of successive targets. Restating these points in the Discussion would strengthen your arguments.On a positive note, reviewer 2 wondered whether the apical dendrites analyzed here belong to thick or slender tufted pyramidal cells in layer 5 because the two types of pyramidal cells significantly differ in function. Your new analysis has revealed their differences in spine density and this reviewer applauded this finding, and also found your quantitative analysis and conclusion that dendritic size is a key factor in determining inhibitory synapse density a strong contribution.We appreciate your comments and have made all changes to the description of synapses as requested:- We start by describing shaft and spine synapses, only (subsection “Putative inhibitory to excitatory synapse ratio”);putative inhibitory after the calibration (subsection “Shaft vs. spine synapses” ff);- Switch to the term - Added a cautionary statement to the beginning of the Results;- Added a paragraph to the Discussion (subsection “Distinction of excitatory vs. inhibitory synapses”).


The ball sports-induced targetoid erythema (SITE) sign is a term that refers to annular erythematous lesions surrounding normal skin that occur after ball to skin contact. Associated purpura may be present or absent. A 19-year-old college student presented with multiple purpuric lesions of targetoid appearance. Additional history revealed that the lesions corresponded with the areas of ball contact during a paintball game. Similar lesions have been described in the participants of other sports, including floorball, ping pong, racquetball, and squash. When evaluating a patient with targetoid erythema with or without accompanying purpura, additional history of recent participation is an activity involving a high-velocity ball may be useful for establishing the diagnosis of the ball SITE sign. Dermatoses may be associated with ball sports. One condition that has been observed is the ball sports-induced targetoid erythema (SITE) sign. The lesion may occur in participants of sports in which the size of the ball is usually small and the velocity is high .The ball SITE sign was previously described as sports purpura . HoweverA 19-year-old college student presented for evaluation of multiple asymptomatic red, skin lesions. Cutaneous examination showed three purpuric lesions that had a targetoid appearance of ecchymosis with normal-appearing skin in the center on his left scapula, left anterior shoulder, and left chest  showed partial spontaneous resolution of the purpuric lesions on the left scapula and left anterior shoulder. The left chest lesion had completely resolved.The ball SITE sign has been associated with various sports Table  1-10]. -10. 1-10The weight of the ball can be as low as 2.5 grams for ping pong balls and as high as 58.5 grams for racquetballs. The weight for squash balls can be as high as 25 grams. In contrast, paintballs weigh approximately 3.3 grams ,5,6,9,11The diameters of the balls also vary. Racquetballs, squash balls, and ping pong balls all have greater diameters of approximately 57 millimeters, 40 millimeters, and 39 millimeters, respectively. In contrast, the paintball has a much smaller diameter of only 17 millimeters ,9,11.The speed of the ball is usually fast. Paintballs have been observed to travel at 191 to 205 miles per hour. In contrast, the other balls travel slower than the paintballs. Ping pong balls travel at 100 miles per hour, whereas racquetballs (traveling 77 to 191 miles per hour) and squash balls (traveling greater than 150 miles per hour) often move faster ,9,11. Our patient developed the ball SITE sign following contact with his skin from paintballs. The ball SITE sign occurring in participants receiving skin impact from paintballs has been described previously Table  4-8]. -8. 4-8].What is now referred to as paintball purpura was initially described as paint pellet purpura by Siegel in 1986 -8. The mThe differential diagnosis of ball SITE sign is described in Table The treatment of paintball purpura is symptomatic. They lesions typically resolve spontaneously as they did in our patient. Also, they often reoccur on new sites of skin contact when the individual again participates in the activity. The term ball SITE sign was introduced to characterize lesions of targetoid erythema that present after high-velocity contact of ball to skin; these lesions occur in the setting of various sports, including paintball, ping pong, racquetball, and squash. The annular red ring of erythema may or may not concurrently present with purpura; our patient’s lesions had purpura. Also, similar to our patient, the lesions typically resolve spontaneously; therefore, a proper history is necessary to establish the diagnosis and avoid additional evaluation or unnecessary treatment.


This paper looks at entropy generation during ice slurry flow in straight pipes and typical heat exchanger structures used in refrigeration and air-conditioning technology. A dimensionless relationship was proposed to determine the interdependency between flow velocity and the volume fraction of ice, for which the entropy generation rates were at the minimum level in the case of non-adiabatic ice slurry flow. For pipe flow, the correlation between the minimum entropy generation rate and the overall enhancement efficiency was analyzed. As regards heat exchange processes in heat exchangers, the authors analyzed the relationship between the minimum entropy generation rate and the heat exchange surface area and exchanger efficiency. The mass fraction of ice crystals in the ice slurry, which may be transported in practice, do not exceed 30%. As an environmentally neutral and natural heat carrier, ice slurry demonstrates a refrigeration potential commensurate to that of refrigerants. Ice slurry flow may be accompanied by phase segregation, which leads to a change in the mean density values and in the value of the dynamic coefficient of fluid viscosity, consequently leading to higher flow resistance values h = h/hrefref)1/3 ,32, multfref)1/3 ,36,37,38fref)1/3 ,40 and dfref)1/3 ,46,47,48In the boiling process, an analysis of the entropy generation rate was used, for example, to determine the preferred scope of use of plain and micro-finned tubes  or to opPaper  is a morThe issues concerning entropy generation in thermal and flow processes with ice slurry were discussed in the studies ,39,40,46The entropy generation rate in the heat exchange process involving ice slurries was discussed in , where tAn analysis of the above papers suggests a very broad use of entropy generation rates for the assessment of diverse thermal ,35,38,43Nu−f > 1. The innovative element presented in the paper was proposing a dimensionless relationship to identify the interdependency between the flow velocity and the mass fraction of ice for which the entropy generation rate was at its minimum level.The use of ice slurry in refrigeration processes is a heat transfer enhancement method applied in refrigeration and air-conditioning technology. Ice slurry fed into a heat exchanger is characterized by both mass flux and mass fraction of ice, which determine the heat transfer process intensity and momentum exchange, inducing various entropy generation rates. This article attempts to identify the range of velocity and mass fraction of ice values which ensure the minimum entropy generation rate during the flow of ethanol-based ice slurry, being a non-Newtonian Bingham’s fluid, through straight 0.01–0.02 m pipes. In addition, the study contains aconducted an analysis of the total entropy generation rate in lamelled air coolers and plate fluid coolers in the context of the optimization of the mass fractions of ice and mass fluxes of the ice slurry. In this case, the analysis focused on the relationships between ice slurry’s flow parameters, the heat exchange surface area, the heat exchanger’s efficiency and the total entropy generation rate on the side of the refrigerant and the cooled medium. New aspects presented in the paper refer to a comprehensive approach to thermodynamic analysis of thermal-flow processes with ice slurry, both for flow in straight tubes as well as in heat exchangers. The analysis of minimum total entropy fluxes in heat exchangers allowed to verify the usefulness of the results of the minimization of the entropy flux of ice slurry in the conditions of forced convection of ice slurry in straight tubes. Additionally, the study compares the results of determination of optimal parameters of flow velocity and mass fractions of ice according to the criterion of minimum entropy and the condition of εAssuming a homogenous single-dimensional flow of the ice slurry mass flux tal pipe , the firIn the test section, the ice slurry entropy changes as per Equation (3):Using Equations (1) and (3), Equation (2) can be transformed into (4):Writing the Fourier’s equation for the ice slurry heat transfer process (5) and using the relationship IST corresponded to the mean temperature of the ice slurry, Equation (6) makes it possible to determine the entropy generation rate for a unit length of the pipe. Equation (6) is true for water-based ice slurry with a constant melting point. For ice slurries based, for example, on a 10.6% aquatic solution of ethanol, the temperature glide with complete melting of ice in a 30% ice slurry amounts to 2.2 K. Therefore, it was assumed that in (6), The determination of the entropy generation rate from Equation (6) for non-adiabatic flow of ethanol-based ice slurry requires the identification of both the pressure drop and the heat transfer coefficients for specific heat transfer conditions. The ice slurry under consideration is a Bingham fluid ,19 and iBy using Equation (7), Fanning’s factors for technically plain pipes can be found for the laminar and turbulent flow area on the basis of Equations (8) and (9), respectively:Ultimately, ice slurry flow resistance values are calculated using Equation (10).n* and consistency index K*. For Bingham fluid and an arbitrary geometry of cross-section, these parameters are found from equations included in Equation (7) makes it necessary to determine a characteristic flow-behavior index For the generalized non-adiabatic flow of ice slurry, the heat transfer coefficient may be calculated for laminar and flow areas from Equations (11) and (12), respectively :(11)NuL=iB in Equations (11) and (12) are shown in The constants 11) and 1 are show(11) and  are showsx < 30%. Calculations were made for ice slurry flow through straight pipes with diameters i =d 0.01, 0.016, 0.02 m, mass flux of ice slurry −1 and heat flux density −2. id = 0.016 m pipe with a constant heat flux density −2.The entropy generation rate was studied for forced convection during melting a 10.6% ethanol solution-based ice slurry with a mass fraction of ice of 0 <sx < 10% and the minimum value of the entropy generation rate is recorded in the turbulent flow area. This is related to the mutual relationship between entropy generation in thermal and flow processes  are typically associated with a relatively large range of velocities, for which the total entropy generation rate is at its minimum. This results from the dominant impact of solid particles and phase change, but not flow velocity, on heat transfer coefficients [In the studied range of flow velocities 0 ≤ ficients . In thisThe analysis of Equations (6)–(12) and calculation results presented in s)Sw) using the measured values of heat transfer coefficients and the flow resistance values [s,x the measurement points were chosen at which s)Sw) [on (13)) :(13)ReBCs, di,xw, s, . Correlation (14) defines the relationship between the Reynolds number and dimensionless numbers taking into account the effect of geometric (qXK) and flow parameters  on the generated minimal entropy flux.All analyzed parameters  makes it possible to determine the optimum velocity values for the condition IS calculated as for Bingham’s fluid) and s, wx both for Nu−f = (Nu/Nupt)/(ptf/f)1/3 = 1, for ice slurry heat transfer during ice slurry flow through id = 0.01 and 0.02 mpipes with constant heat flux density values of −2. The range of sx and w values, for which εNu−f > 1, means that while the same power is required to transport the refrigerant, the use of ice slurry over the use of the carrier fluid as the refrigerant translates into better thermal efficiency. The results shown in id = 0.01 m, for which entropy generation minimization implies lower values of flow velocity with respect to the condition εNu−f > 1. sx and w. In design practice, the recommended flow velocities in pipes amount to w < 1 ms−1. This velocity range for the flow of ice slurry in pipes with id < 0.02 m enables effective enhancement of the heat transfer process with the minimum entropy generation rates.○w < 2 ms−1.During pipe flow, the lowest entropy generation rates were characteristic of small mass fractions of ice in the turbulent flow area and for the flow velocity of 1.5 < ○sx = 30%.In the laminar flow area, the lowest entropy generation rates corresponded to the highest analyzed mass fraction of ice ○−2 are greater than w > 1 ms−1.Regardless of the share of solid particles, the minimum entropy generation rate criterion requires the application of high flow velocities, which for heat flux density values of The calculations performed suggest that:An analysis of the entropy generation rate in heat exchangers was performed under conditions which differ from those present during flow through pipes. For pipe flow, a specific heat flux density, flow velocity and mean mass share of ice are assumed for which the entropy generation rates are calculated. For heat exchangers, the exchanger’s thermal efficiency, ice slurry mass flux and the inlet value of the mass fraction of ice are assumed. A change in ice slurry enthalpy, the heat exchange surface (heat flux density) and the end temperature of the cooled medium result from the balance equations and, as a consequence, make it possible to determine the entropy generation rate.The heat transfer process enhancement, greater specific enthalpy of the ice slurry and the almost constant melting point enable ice slurry-fed heat exchangers to have a smaller heat exchanger surface than heat exchangers which rely on single-phase refrigerants. The heat exchange surface area has an impact, for example, on the values of flow resistance in the heat exchanger. In this part of the article, the authors discussed the impact of the ice slurry mass flux and the mass fraction of ice on the change in the total entropy generation rate in heat exchangers. To achieve this, the heat transfer process was discussed for two types of heat exchangers: A lamelled air cooler and a plate heat exchanger, which was used as a milk cooler.Assuming that the melting process of the ice slurry occurs at a constant temperature, it is possible, on the basis of , to exprTaking into consideration the ice melting process in the ice slurry, Equation (17) may be converted into (18)If the fluid flow within the heat exchanger is treated as quasi-adiabatic flow, we can state thata-outp = 1 bar) was assumed. On the other hand, air pressure at the inlet to the exchanger was a-outp = a-outp + Δap.Equation (20), following integration, leads to Equation (21):In order to determine the entropy generation rate in the lamelled air cooler, a calculation algorithm was prepared based on Peclet’s equation and balance equations for the air and the ice slurry . The air○constant thermal efficiency values ○−1,constant air flow stream ○a-inT = 20 °C,constant inlet air temperature ○−1,various ice slurry mass flux values 0.3 ○sx ≤ 30%,various mass fractions of ice 5 ≤ ○id = 0.01 m pipes.constant number of pipes and feeds in the heat exchanger made of The calculations were made for:−1). This results in a significant increase in entropy generation associated with ice slurry and air-side flow resistances. The Bejan number for a 40 kW cooler efficiency changed from 0.04 to 0.43 for the mass fractions of ice of sx = 30% and sx = 5%, respectively. s = 30%. For a constant mass flux of the ice slurry, the dominant entropy generation component is the entropy generated by the air-side heat transfer the air-side heat transfer process. Lower mass fractions correspond to higher mean temperatures of the ice slurry. Therefore, the receipt of the same heat flux is conditional upon a larger change in the specific enthalpy of the refrigerant. The entropy generation rate as a function of the mass flux is not a monotonic function. This has been illustrated by The result of the calculations were different heat exchange surfaces (different pipe lengths), which implied different entropy generation rates caused by the flow resistances of the refrigerant and air. A change in the entropy generation rate also resulted from variable mass fluxes of the ice slurry and changes in the specific enthalpy of the ice slurry. sx = 30% indicate that for the discussed structure of the exchanger, function −1. At the point of the minimum of function Calculations performed for a constant thermal efficiency of id = 0.01 m pipe. Just as in the case of flow through a straight pipe, in the discussed heat exchanger higher heat flux densities also corresponded to higher optimum flow velocities. The comparison :iB for a rectangular cross-section, included in In order to determine the entropy generation rate in a plate heat exchanger/fluid (milk) cooler, an algorithm  was deve○constant thermal efficiency values ○f−inT = 12, 20 °C and f−inT = 2, 5 °C ,a change in the temperature of the fluid cooled in the exchanger between ○−1,various ice slurry mass flux values 0.3○sx ≤ 30%,various mass fractions of ice 5 ≤ ○pn = 6, sn = 1; pn = 12, sn = 2.two geometrical configurations of the exchanger: The calculations were made for:pn = 6, sn = 1). In this calculation variant, the constant mass flux condition implies variable values of changes in the ice slurry’s specific enthalpy and various degrees of ice melting. Just as in the case of the air cooler, regardless of the mass flux, minimum entropy generation rates were recorded for the maximum mass share of ice sx = 30%. For a constant mass flux of the ice slurry, the dominant component of entropy generation is that generated by the ice slurry-side heat transfer process. In a plate heat exchanger, the entropy generation due to flow resistances is determined by the flow of the ice slurry. In the calculations performed by the authors, the flow was of a laminar nature and the greatest flow resistances were generated for an ice slurry with the mass share of ice of sx = 30%, for which the dynamic plastic viscosity coefficient and limit shear stress were highest. The highest Bejan number of 0.15 corresponded to sx = 30%, −1.pn = 12, sn = 2) and a constant mass share of ice sx = 30%. Function K = 380−530) was recorded for laminar flow, but for w = 1.8−2.3 ms−1. These flow velocity values are significantly higher than the velocity values used in typical plate exchangers (w < 0.5 ms−1). The application of a flow velocity of w < 1 ms−1 leads to an increase in the entropy generation rate by at least 14%–67%, depending on thermal efficiency.−1), lower entropy generation rates are recorded in an ethanol-fed exchanger than in an ice slurry-fed exchanger. If ethanol is used, however, the required surface area of the exchanger is at least 60% higher than in the case of an ice slurry fed exchanger.The curves presented in Using the results of experimental studies and formula for the calculation of flow resistance and heat transfer coefficient values during the melting of ice slurry under forced convection conditions, an analysis of the entropy generation rates was performed during flow in straight pipes and heat exchangers.sw, x) for different tube diameters and heat flux densities for which the entropy flux generated during the melting process in the conditions of forced convection of ice slurry is minimal. The original aspect presented in the paper is the description of the set of optimal parameters for s,w, x the criteria relationship between the Reynolds number and dimensionless numbers taking into account the effect of geometric and flow parameters on the generated entropy flux.The study determined the area of optimal parameters for ethanol ice slurry .Unlike the criterion of minimum entropy flux of ice slurry in a straight channel, the criterion of minimum entropy flux in a heat exchanger always prefers the use of maximum mass fractions of ice. In the first case, high ice mass fractions minimize the entropy flux only for low flow velocities (


Tauopathies are a class of neurodegenerative disorders characterized by abnormal deposition of post-translationally modified tau protein in the human brain. Tauopathies are associated with Alzheimer’s disease (AD), chronic traumatic encephalopathy (CTE), and other diseases. Hyperphosphorylation increases tau tendency to aggregate and form neurofibrillary tangles (NFT), a pathological hallmark of AD. In this study, okadaic acid , a protein phosphatase 1/2A inhibitor, was treated for 24h in mouse neuroblastoma (N2a) and differentiated rat primary neuronal cortical cell cultures (CTX) to induce tau-hyperphosphorylation and oligomerization as a cell-based tauopathy model. Following the treatments, the effectiveness of different kinase inhibitors was assessed using the tauopathy-relevant tau antibodies through tau-immunoblotting, including the sites: pSer202/pThr205 (AT8), pThr181 (AT270), pSer202 (CP13), pSer396/pSer404 (PHF-1), and pThr231 (RZ3). OA-treated samples induced tau phosphorylation and oligomerization at all tested epitopes, forming a monomeric band (46–67 kDa) and oligomeric bands (170 kDa and 240 kDa). We found that TBB (a casein kinase II inhibitor), AR and LiCl (GSK-3 inhibitors), cyclosporin A , and Saracatinib (Fyn kinase inhibitor) caused robust inhibition of OA-induced monomeric and oligomeric p-tau in both N2a and CTX culture. Additionally, a cyclin-dependent kinase 5 inhibitor (Roscovitine) and a calcium chelator (EGTA) showed contrasting results between the two neuronal cultures. This study provides a comprehensive view of potential drug candidates , and their efficacy against tau hyperphosphorylation and oligomerization processes. These findings warrant further experimentation, possibly including animal models of tauopathies, which may provide a putative Neurotherapy for AD, CTE, and other forms of tauopathy-induced neurodegenerative diseases. Tauopathy is a class of neurodegenerative conditions that are associated with pathological phosphorylated tau protein accumulation in the human brain. Tauopathy has been associated with several clinicopathological conditions, including chronic traumatic encephalopathy (CTE) , traumatTau can be subjected to a sequence of post-translational modifications that may attenuate the protein’s structure, function, turnover, or can lead to multimeric aggregation. In addition to phosphorylation, these modifications include acetylation , methyla2+/calmodulin-dependent protein kinase II (CaMKII); cyclin-dependent kinase 5 (CDK5); casein kinase (I/II); dual-specificity tyrosine kinase phosphorylation and regulated kinase-1A/2 (DYRK1A/2), glycogen synthase-3 (GSK-3), and tyrosine kinase Fyn pyrrolo-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (Saracatinib) , -6-methoxy-5-methyl-7-(methylamino)-6,7,8,9,15,16-hexahydro-17-oxa-4b,9a,15-triaza-5,9-methanodibenzocyclonona[jkl]cyclopenta[e]-as-indacen-14(5h)-one (STS) , Z-Asp-2,6-Dichlorobenzoyloxymethyl Ketone (Z-DCB)  and okadaic acid . SNJ-1945 was a gift from  .Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid : AT8  , 83, AT2® CCL-131™, Manassas, VA, USA) and were grown as recommended by the manufacturer [ºC in a humidified 5% CO2-containing atmosphere.Mouse neuroblastoma N2a cell culture were purchased directly from American Type Culture Collection company , supplemented with 1% B-27 (Thermo Fisher), one mM Glutamine (Thermo Fisher), and incubated at 37°C in a humidified 5% CO2-containing atmosphere. The medium was replaced every three days.Established rat primary cerebrocortical neuronal culture  harvested from a homogenized pool of day ten Sprague–Dawley rat brains and plated on poly-L-lysine-coated (0.01% (w/v)) 12-well culture plates , similar to previously described methods  at a denTable 1).In all experimental conditions, endogenous tau was used to asses the tau phosphorylation levels. For N2a cell culture treatments, complete media were replaced with serum-free DMEM media. For CTX primary cultures, all experiments were performed after ten days in culture, and the media was replaced with Neurobasal® media supplemented with 0.5% B-27. For both CTX and N2a culture, SNJ-1945  and Z-DCB  were added to all experimental conditions before the treatment for 1h. Next, okadaic acid  was added for 24h followed by protein kinase inhibitors for 6h. The protein kinase inhibitors used included: K252a (10 μM), AR-A014418 (60 μM), A-1070722 (60 μM), Saracatinib (100 μM), LiCl (5 mM) TBB (30 μM), EGTA (five mM), Roscovitine (60 μM), STS (0.5 μM), CsA (60 μM) (if added) . The Triton-X lysis buffer included: 1mM DTT, 1% phosphatase inhibitors (Sigma), 1% Mini-Complete protease inhibitor cocktail tablet , and 1% Triton X-100. The attached cells were then scraped down in the lysis buffer and collected into a separate 1.5 ml Eppendorf tube. The insoluble pellets from the conditioned culture media were combined with the lysed cells in the lysis buffer. The cell lysates were incubated for 90 minutes at 4Protein concentrations of cell lysates were determined by bicinchoninic acid microprotein assays  against albumin standards. Equal protein samples (20 μg) were prepared for SDS–PAGE in 8x loading buffer containing 0.25 M Tris (pH 6.8), two mM DTT, 8% SDS, and 0.02% bromophenol blue. Each sample was subjected to SDS–PAGE electrophoresis on a 4–20% precast-gels (Bio-Rad) and then transferred onto PVDF membranes. The membranes were blocked in 5% milk for 1h and then incubated with primary antibodies (1/1000) overnight. The secondary antibodies, anti-rabbit or anti-mouse IgG conjugated phosphatase, were then added for 1h at room temperature afterward. The blots were then washed with TBST, and immunoreactive bands were visualized by developing with biotin, avidin-conjugated alkaline phosphatase, Nitro blue tetrazolium, and 5-Bromo-4-chloro- 3-indolyl phosphate (BCIT/NBT) developer . A 250 kDa to 14 kDa rainbow molecular weight marker  was loaded in the first well of the electrophoretic gel to estimate the molecular weight of each band. Quantitative evaluation of protein levels was performed via computer-assisted densitometric scanning .All experiments were done independently on different days at least three times. N2a cells were seeded at least three separate times, and each experiment was treated independently with the chemicals accordingly. As for the primary culture, three separate batches of n = 3  were obtained from the company on different days. All biological replicates treatments were done at different times and days. Data are plotted as mean ± SEM. Statistical analysis was performed with one-way ANOVA Tukey’s Test. For multiple comparisons, one-way ANOVA followed by Bonferroni’s post-hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, **** p<0.0001, ns: non-significant. GraphPad Prism 8.0 .Fig 1A and 1B). OA concentration was selected based on previous studies that used similar concentrations optimized on neuronal cell culture [Okadaic acid (OA), a potent PP2A/PP1 inhibitor, induces tau hyperphosphorylation and aggregation , 91. To  culture , 91–93.  culture , cell-pe culture , 96. To  culture . Cleavag culture , 98.. Western blots were analyzed with total tau monoclonal antibody DA9 (a.a. 102–140), and monoclonal phospho-tau (p-tau) antibodies, including CP13 (pSer202), and PHF-1 (pSer396/pSer404) (Table 1). The β-actin antibody was used to evaluate the evenness of loading the protein extracts. Untreated control showed that the total tau antibody DA9 detected tau protein bands at 46 kDa and 48 kDa . The intensity of the band at 46 kDa was higher compared to the band at 48 kDa in control samples .Control samples probed with αII-spectrin showed a high molecular weight 240 kDa band (intact αII-spectrin), while SBDPs were absent, suggesting a healthy metabolism and neuronal culture Fig 1A and 1B).Treatment with OA (100 nM) for 6h and 24h showed a dramatic decrease in levels of the 46 kDa and increased levels of 48 kDa with DA9 antibody. The 46 kDa and 48 kDa bands may be different tau isoforms, as reported in previous studies . These b. However, with OA treatment, CP13 showed a high molecular weight (HMW) band formed at 110 kDa (x2 size of monomeric tau) with 6h and 24h . Probing with the PHF-1 antibody (pSer396/pSer404) did not show any tau band with control samples . Treatment with OA for 6h and 24h showed HMW cluster of bands at 220 kDa, 240 kDa, and 260 kDa with PHF-1 . Notably, the 260 kDa band (red arrow)  was only detectable with OA treatment for 24h (PHF-1). Low molecular weight monomeric tau (LMW-MT) bands were not detected with either CP13 or PHF-1. It should be noted that the DA9 antibody recognizes total tau epitopes from aa. 102–140. Thus, to identify the 48 kDa tau species detected with DA9, the phospho-tau antibody needs to recognize the same epitope. It possible that LMW-MT might be phosphorylated at sites other than Ser202/Ser396/Ser404, and oligomerized into the different HMW tau species detected at 110 kDa, 170 kDa, 220 kDa, 240 kDa, and 260 kDa. Using RZ3(pThr231) and AT270(pThr181), LMW-MT at 48 kDa, and 55 kDa were detected with OA treatment, respectively , pSer396/pSer404 , RZ3  and AT270 .Probing with CP13 (pSer202) antibody did not show any detectable bands of tau protein in control samples  All conditions were pretreated with SNJ-1945  and Z-DCB  to minimize apoptotic pathway activation  [Table 1).To screen for protein kinase inhibitors as drug candidates for inhibition of OA-induced hyperphosphorylation and oligomerization, mouse neuroblastoma N2a cells were pretreated with OA for 24h followed by treatment with protein kinase inhibitors for 6h. The positive control included only OA treated cells for 24h. Protein kinase inhibitors used included: LiCl (10 mM), AR-A014418 (AR) (60 μM), A-1070722 (A107)(60 μM), K252a (10 μM), STS (0.5 μM) 4,5,6,7-tetrabromobenzotriazole (TBB) (60 μM), Roscovitine (60 μM), Saracatinib 100 μM), cyclosporine A (CsA) (60 μM), and EGTA (5 mM) (eolysis) , 100 . CP13 (pSer202) antibody showed that TBB eliminated the OA-induced 110 kDa band (oligomeric p-tau). Similarly, the PHF-1 antibody (pSer396/pSer404) showed that TBB fully inhibited the formation of 240 kDa (HMW bands-oligomeric tau) (Table 3). As a selective casein kinase II (CKII) inhibitor, TBB showed robustness in inhibiting both OA-induced tau hyperphosphorylation and oligomerization. Thus, the aim was to evaluate the TBB dose-response effect on N2a neuronal culture. Thus, N2a cells were treated with OA for 24h followed by treatment with various concentrations of TBB  for 6h . The result shows that treatment with ten micromolars of TBB resulted in a 50% reduction of the 110 kDa , 48 kDa, and 170 kDa bands  . Increasing the concentration of TBB up to 30 μM caused a 90% reduction of 48 kDa , 170 kDa  and 110 kDa  . As for assessing neuronal culture integrity, the intact αII-spectrin band was detected at 240 kDa, and no SBDP150/145 or SBDP120 was observed with the TBB treated conditions suggesting a healthy culture.Since aberrant CKII has been reported in AD , TBB, a S1 Fig). Moreover, CsA reduced the p-tau band at 110 kDa of CP13 , and 240 kDa of PHF-1 (oligomeric p-tau band) (Table 3).Cyclosporin A (CsA) has been reported to inhibit the calcineurin phosphatase activity (PP3) and CaMKII by blocking the Ca++ mitochondrial permeability  Table 1Table 1. S1 Fig). As for the AT270 antibody, OA-treated samples showed a band detected at 55 kDa (monomeric p-tau), which was abolished when neurons were treated with CsA. The 48 kDa and 55 kDa OA-induced bands detected by RZ3 and AT270, respectively, indicate that tau can have various levels of phosphorylation, depending upon the epitope tested. β-actin protein levels remained even in all experimental conditions. Blotting with the αII -spectrin antibody did not show any significant changes of the 240 kDa band (intact form). Moreover, SBDP150/145 or SBDP120 immunoreactive bands were not detected in all of the treated samples, indicative of a healthy metabolism.RZ3 antibody showed a complete reduction of the 48 kDa band (monomeric p-tau) when cells were treated with CsA . EGTA caused a 90% reduction of the 110 kDa band  and an 85% reduction of 240 kDa  (Table 3). As for apoptotic pathway activation, αII-spectrin antibody did not show any effect on the 240 kDa band (intact form).Another calcium-dependent kinase inhibitor, EGTA, was used as a calcium-chelating agent. EGTA has a lower binding affinity for Mga++ ions . Total tTable 3). As for total tau DA9 antibody, LiCl also showed an opposite effect by increasing the 48/46 kDa (p-tau/non-phospho-tau) band ratio by 20%, and the 170 kDa oligomeric tau band by 12%.The effects of small molecule GSK-3 inhibitors, LiCl, A-1070722 (abbreviated as A-107), and AR-1014418 (abbreviated as AR), were tested on OA-induced tau hyperphosphorylation and oligomerization. LiCl showed an opposite effect in N2a cell treatment by increasing levels of 110 kDa band  and levels of 240 kDa band  (Table 3). Probing with total tau DA9 showed that AR treatment caused an adverse effect by increasing the 48 kDa/46 kDa ratio  and reducing the 170 kDa oligomeric form band by 36% (Table 3). Another potent GSK-3 inhibitor, A-107 (Ki = 0.6 nM for GSKα and GSK-3β) [Table 3). Probing with total tau DA9 showed with A-107 treatment, a 13% reduction of the 170 kDa band (DA9), and did not show a statistically significant effect on the 48 kDa band (Table 3). As for caspase-3, calpain, and cell injury activation, αII-spectrin did not show SBDP 150/145 or SBDP120 post-treatment, indicative of a healthy neuronal culture.Treatment with AR did not show a statistically significant effect on the 240 kDa band  or 110 kDa band  ( GSK-3β) , was selTable 3). Saracatinib treatment reduced (p<0.0001) the 110 kDa oligomeric p-tau band of CP13  and produced a 46% reduction in immunoreactivity of the 240 kDa (oligomeric p-tau form band of PHF-1) (Table 3). As for assessing cell integrity, intact spectrin (240 kDa) levels remained constant, and SBDP150/145 and SBDP120 levels were not significantly altered.In the current study, Saracatinib was selected to investigate the role of the Fyn kinase function on the tauopathy-relevant cell-based model . Probing50 = 32.9 nM), Ca2+/calmodulin-stimulated phosphodiesterases (IC50 = 1.3–2.9 μM), serine/threonine protein kinases (IC50 = 10–20 nM), myosin light-chain kinase , receptor tyrosine kinases, and inhibiting the carcinogenic properties of MET oncogene [Table 3). K252a treatment caused a 40% reduction of 170 kDa  compared to OA treatment alone (Table 3). For p-tau detection, probing with the CP13 antibody showed a 60% and a 32% reduction in the 110 kDa (oligomeric p-tau form) with K252a and STS treatment correspondingly. PHF-1 showed a 70% and 80% reduction in levels of 240 kDa (oligomeric form) with K252a and STS treatment, respectively (Table 3). αII-spectrin immunoreactive bands  did not show a statistically significant difference compared to control values.K252a is a non-selective cell-permeable protein kinase inhibitor, inhibiting protein kinase C . Roscovitine showed partial, but a statistically non-significant decrease in the 48 kDa (monomeric p-tau) band of DA9, compared to OA treatment alone. The αII-spectrin antibody did not show a statistically significant difference of intact form (240 kDa), SBDP150/145, and SBDP120 compared to control values.Roscovitine is a cyclin-dependent kinase 5 (CDK5) inhibitor that acts through direct competition at the ATP-binding site . RoscoviTable 1) followed by treatment with protein kinase inhibitors for 6h. Calpain and caspase-3 inhibitors, SNJ-1945 and Z-DCB, respectively, were added to all experimental conditions to prevent cell death-mediated proteolysis of tau as a potential confound. culture health.To further expand our experimental paradigm in a culture-based model suitable for drug candidate screening, the effectiveness of the protein kinase inhibitors was investigated on differentiated rat primary cerebrocortical neuronal (CTX) cultures. Therefore, CTX cells were pretreated with or without OA 24h (100 nM) . Treatment with OA for 24h caused a dramatic increase of 67 kDa band (monomeric p-tau) at multiple phospho-tau epitopes  .CTX control cultures showed normal cell bodies and healthy neurites, including axons and dendrites. Untreated control samples showed basal levels of phosphorylated tau (67 kDa) detected by total and p-tau antibodies, including: DA31 (a.a.150-190), CP13 (pSer202), RZ3 (pThr231), PHF-1 (pSer396/pSer404), AT8 (pSer202/pThr205), and AT270 (pThr205) (Table 4). Total tau DA31 (a.a. 150–190) antibody detected immunoreactive bands at 63 kDa and 67 kDa with the different kinase inhibitor treatments. The decreased electrophoretic mobility of the 63 kDa might correspond to the lower levels of p-tau protein induced by the protein kinase inhibitors; thus, this band was assigned as non-phospho-tau. TBB treatment reduced the phospho-tau band at 67 kDa , and increased non-phospho tau band at 63 kDa  (Table 4).Since the concentration of 30 μM TBB resulted in at least 90% inhibition in N2a cells, the same concentration was used for CTX culture. Treating CTX culture with TBB reduced basal and OA-induced tau phosphorylation (67 kDa) at CP13 , RZ3 , PHF-1 , AT8 , and AT270  compared to OA treatment alone . With total tau DA31 (a.a. 150–190), LiCl and AR eliminated the 67 kDa (monomeric p-tau) and substantially increased the 63 kDa band (non-phospho tau). Treating CTX neuronal culture with A107 also showed a substantial inhibition of 67 kDa (-OA and +OA) band with CP13 , RZ3 , PHF-1 , AT8 , AT270 , and total tau DA31 , compared to OA treatment alone (Table 4). As for Roscovitine treatment, in contrast to N2a neuronal treatment, the 67 kDa band was reduced considerably at CP13 , RZ3 , PHF-1 , and total tau DA31 . However, Roscovitine did not show a statistically significant effect on OA-induced tau phosphorylation at AT8 and AT270 (Table 4).In contrast to N2a cells, LiCl reduced basal and OA-induced tau phosphorylation  in CTX culture at CP13 , RZ3 , PHF-1 , AT8 , AT270  and total tau DA31  . Regarding the 67 kDa band, CsA had dramatic inhibition on basal tau phosphorylation at: CP13 (90%), RZ3 (91%), PHF-1 (89%), AT8 (95%), AT270 (92%) and total tau DA31  (Table 4). With OA treatment, CsA also showed a considerable immunoreactivity reduction of the 67 kDa band at the epitopes: CP13 (33%), AT8 (86%), and total tau DA31 (28%). CsA had no effect on 67 kDa band at PHF-1, AT270, and RZ3 compared to OA treatment alone (Table 4). Minor oligomeric bands were observed at 240 kDa with PHF-1 antibody in OA treated samples. Based on the αII-spectrin blot, CTX cultures demonstrated intact spectrin (240 kDa) and the absence of any SBDPs, suggesting a healthy metabolism under the experimental conditions. Furthermore, Saracatinib reduced basal and OA-induced tau hyperphosphorylation at: CP13 , RZ3 , PHF-1 , AT270  and total tau DA31 . Saracatinib did not show any significant effect at AT8 phospho-tau epitope (pSer202/pThr205 sites) , RZ3 (pThr231), and DA31 (a.a. 102–145), presumably accounting for the dephosphorylation of tau (Table 4). K252a did not show any statistically significant inhibition at AT8 and AT270 with both basal and OA-induced tau hyperphosphorylation (Table 4). Cultures treated with STS showed considerable reduction of basal and OA-induced tau phosphorylation at CP13 , RZ3 , PHF-1 , AT8 , AT270 , and total tau DA31  (Table 4). Unexpectedly, EGTA caused an adverse effect in CTX culture by further enhancing physiological p-tau and OA-induced tau hyperphosphorylation at CP13 , RZ3 , PHF-1 , AT8 , AT270 , and total tau DA31  (Table 4).Treatment with K252a caused substantial inhibition of the 67 kDa band, with basal and OA-induced treatments at CP13 , RZ3 , PHF-1 , and total tau DA31   as a model to screen for various tau kinase inhibitors as potential drug candidates. The N2a neuronal cultures have been widely used to study mechanisms of neurodegeneration because they are a homogenous culture system convenient to handle and can multiply quickly to produce a tremendous amount of neuron precursor cells [in vivo expression. Additionally, primary culture has the advantage in portraying the complexity of the central nervous system by better translating into in vivo models used for screening pharmaceutical drug candidate compounds [It has been reported that OA results in robust tau hyperphosphorylation at multiple pathological epitopes in animal and cell culture studies , 106–108or cells . Our CTXompounds . Thus, t showed a faint band at 48 kDa and a major band at 46 kDa. Upon the addition of OA to N2a culture, we observed a significant increase in the 48 kDa band and a decrease in the 46 kDa. Both bands are within the range of monomeric tau protein; however, the fact that OA addition caused this shift suggests that these two different bands might not represent only spliced variants of tau protein. It would be expected to observe an increase in both band's phosphorylation levels after OA treatment, even if they were alternatively spliced form of tau.Western blot analysis showed that OA induced tau hyperphosphorylation and oligomerization at various phospho-tau epitopes in N2a cell culture as a tauopathy model. It is well-known that OA induces apoptosis in human neuroblastoma cells, mouse neuroblastoma, and rat cerebellum neurons . Thus, iFigs  and 5 might be a different tau isoform expressed from the ones in N2a cell culture. Several studies have analyzed the alternatively spliced tau protein using western blotting, showing a distinct profile of molecular weights ranging from 46–67 kDa corresponding to the isoforms 0N3R, 0N4R, 1N3R, 2N3R, 2N4R [ might be a product of p-tau self-aggregation, including dimers and trimers. It was discovered that any tau isoforms could develop into tau dimers. Two independent tau proteins can associate through cysteine residues via microtubule-binding domain (MBD), creating tau dimers linked covalently by a disulfide bridge, and organized in an anti-parallel direction [Moreover, the 63–67 kDa band detected in CTX culture 3R, 2N4R –114. As 3R, 2N4R . The higirection . There airection . The molirection –119. Tauirection . Furtherirection . Althougirection . Thus, iirection . These p, the reduction of the non-OA induced p-tau by the kinase inhibitors indicate the involvement of the several kinases under physiological conditions.Previous reports using immunocytochemistry and western blotting showed that in CTX culture, tau is physiologically highly phosphorylated . Our CTXIn contrast to N2a cell culture, only the monomeric form of tau (ranging from 63 kDa– 67 kDa) was observed in CTX culture. One possible reason for such an effect would be that our separation of tau protein by SDS-PAGE was carried out under reducing conditions (Dithiothreitol (DTT) and β-ME) that could minimize tau oligomers to the monomeric form in CTX primary culture. On the other hand, our CTX serum-free neurobasal media contained antioxidants such as glutathione. Thus, the addition of these antioxidants may have blocked the process of tau oligomerization from occurring.50 = 900 nM for rat liver) [In the N2a and CTX neuronal culture, TBB (CKII inhibitor) provided the most profound reversal of tau phosphorylation and oligomerization at the epitopes pSer202, (CP13), pSer396/pSer404 (PHF-1), pSer202/pThr205 (AT8), pThr181 (AT8), and pThr231 (RZ3). TBB is a selective, cell-permeable, ATP/GTP-competitive inhibitor of casein kinase II (CKII) (ICt liver) . CKII fut liver) . CKII cat liver) , 124–126t liver) . Thus, iin vitro and in vivo and has been proposed as a target for pharmacological intervention [ and was less effective in N2a cells . The effect of AR was more prominent compared to another GSK3 inhibitor, A-107, in CTX primary culture. This effect could be attributed, in part, to the high selectivity and specificity of AR to GSK3β [i = 0.6 nM for both) [Since GSK-3 is a well-known kinase that can phosphorylate tau rvention , 45, thrrvention . In our to GSK3β  comparedor both)  thereby or both) . Similaror both) . In anotor both) . The lac2+, but not competitive to the substrate or ATP. Another theory proposes that LiCl causes potassium deprivation [LiCl is well-known to inhibit GSK3 and other kinases . In CTX rivation .The use of CDK5 inhibitor Roscovitine in CTX culture substantially reduced basal and OA-induced tau hyperphosphorylation at CP13 (pSer202), RZ3  and AT270 (pThr181). Roscovitine reduced basal phosphorylation at AT8 (pSer202/pThr205) but did not affect the OA-induced tau hyperphosphorylation, reflecting its specificity and the selectivity to our cell models. Similarly, several recent studies revealed that inhibiting CDK5 with Roscovitine had neuroprotective properties against neurodegenerative conditions caused by decreasing tau phosphorylation , 133. Li [Another protein kinase that has recently received consideration as a pharmaceutical target is the tyrosine kinase Fyn, which has been linked with the amyloid pathway and tau phosphorylation through the N-terminal domain in dendrites . Saracat . In our , Table 2. Several findings have shown that calcineurin inhibition increases tau hyperphosphorylation, and cells treated with CsA could induce the process [, Table 2.Cyclosporine (CsA) or FK506 is an 11 amino acid cyclic non-ribosomal peptide used as an immunosuppressant. CsA induces neuroprotective properties through inhibiting specifically enzyme activity by binding to cyclophilin, forming a complex that inhibits calcineurin (PP3)   and other prospects that have been minimally studied in application to potential therapies (TBB and CsA). TBB and CsA warrant further test design involving an animal model of tauopathy. Exploration of agents that inhibit tauopathy progression is important as recent studies implicate pre-fibrillar hyperphosphorylated tau as the toxic species in AD, CTE, and other neurodegenerative diseases, therefore, re-establishing the interest in tau kinase inhibitors development at putative neurotherapies, which could translate into human clinical trials.S1 FigFig 2 was used to test CsA in N2a cell culture. Twenty micrograms of protein extract were used for the analysis of tau. Calpain and caspase-3 inhibitors (S+Z) were added to all experimental conditions, including the control samples. CsA is known to inhibit the phosphatase activity of calcineurin (PP3). In the presented experiment, it is used to assess its kinase inhibition potential on the monomeric and oligomeric p-tau induced by OA. (a). Immunoblots of N2a neuronal culture protein extracts showing antibodies directed against major tau phosphorylation sites. Two additional p-tau antibodies were used (AT270 and RZ3) to assess the phosphorylation sites at pThr181 and pThr231, respectively. RZ3 and AT270 detected distinctive monomeric p-tau bands at 48 kDa, and 55 kDa, respectively. Total tau levels were probed using DA9 (a.a. 102–145) in N2a cells. Blue colored labels correspond to monomeric or oligomeric p-tau species. Immunoblots were probed with αII-spectrin antibody to monitor calpain and caspase-3 mediated proteolysis. (b). Immunoblots quantification of N2a. The ratio of phosphorylation epitopes levels over β-actin levels ± SD are represented as a percentage of control. n = 3 per condition. For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post-hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: non-significant.The same experimental design mentioned in (PDF)Click here for additional data file.S2 FigA continuation of (PDF)Click here for additional data file.S3 FigFig 4 and Fig 5 legends. Primary neuronal cultures (CTX) were fully differentiated and had healthy neurites when examined under the microscope. All wells were pretreated with S+Z for 1h. For conditions that did not include OA, cultures were treated with CsA for 6h. For OA-induced conditions, OA was added for 24h followed by CsA for 6h. A reverse-time course was followed, and all experimental conditions were collected and analyzed at the same time. Twenty micrograms of CTX culture extracts were run on SDS-PAGE, followed by western blotting. (a). Immunoblots of CTX culture protein extracts. CTX culture using antibodies directed against major tau phosphorylation sites including: CP13 (pSer202), PHF-1 (pSer396/pSer404), RZ3 (pThr231), AT8 (pSer202/pThr205), AT270 (pThr181). Total tau levels were probed using DA31 (a.a. 102–145). The 67 kDa assigned as a monomeric p-tau band, and the 63 kDa band was assigned as monomeric non-phospho tau at the different studied epitopes. (b). Immunoblots quantification. The ratio of phosphorylation epitopes levels over β-actin levels ± SD are represented as a percentage of control. n = 3 per condition. For multiple comparisons, one-way ANOVA followed by Bonferroni’s post-hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: non-significant.The experimental procedures were followed, as described in (PDF)Click here for additional data file.S1 Raw ImagesMW, GE Healthcare rainbow full range molecular weight marker.(PDF)Click here for additional data file.