Sholl Analysis Research Articles

Melatonin ameliorates spatial memory and motor deficits via preserving the integrity of cortical and hippocampal dendritic spine morphology in mice with neurotrauma.

We aimed to elucidate the role of cortical and hippocampal dendritic spines on neurological deficits associated with hippocampal microgliosis, hippocampal neurogenesis, and neuroinflammation in mice with cortical compact impact (CCI) injury. In the present study, we found that CCI reduced spatial memory mean latency (10s. vs 50s) and motor dysfunction (130s. vs 150s.) in mice, as determined by Morris water maze and rotarod test, respectively. Golgi staining of cortical pyramidal neurons revealed that, compared to the controls, the CCI group treated with vehicle solution had significantly lower values of dendritic order (or dendritic branch number) (4.0 vs 6.2), total spine length (400μm vs 620μm) and spine density (40spines/μm vs 60spines/μm), but had significantly higher values of dendritic beading (40beadings/mm vs 20beadings/mm). Additionally, Sholl analysis showed that, compared to controls, the CCI + NS group mice had significantly lower values of dendritic intersections (1.0 vs 2.0). Immunofluorescence assay also revealed that, compared to controls, the CCI + NS group mice had significantly higher values of the newly formed hippocampal cells (1250/mm2 vs 1000/mm2) but significantly lower values of dendritic order (2.0 branch # vs 4.2 branch #), total spine length (180μm vs 320μm) and intersection (1.0 vs 3.0). The CCI + NS group mice further showed significantly higher numbers of microglia in the dentate gyrus of the hippocampus and higher concentrations of pro-inflammatory cytokines in the cerebrospinal fluids. All the CCI-induced spatial memory (40s) and motor (150s) dysfunction, deranged dendritic and spine morphology of cortical pyramidal neurons or hippocampal newly formed cells, hippocampal microgliosis, and central neuroinflammation were all significantly reduced by melatonin administration during post-CCI. Simultaneously, melatonin therapy caused an enhancement in the compensatory hippocampal neurogenesis and neurotrophic growth factors (e.g., doublecortin-1) and compensatory central anti-inflammatory cytokines. Our results indicate that melatonin attenuates the spatial memory and motor deficits via the modification of cortical and hippocampal dendritic spine morphology, hippocampal microgliosis and neurogenesis, and neuroinflammation in mice with traumatic brain injury.

Disrupted Coordination of Hypoglossal Motor Control in a Mouse Model of Pediatric Dysphagia in DiGeorge/22q11.2 Deletion Syndrome.

We asked whether the physiological and morphologic properties of hypoglossal motor neurons (CNXII MNs) that innervate protruder or retractor tongue muscles are disrupted in neonatal LgDel mice that carry a heterozygous deletion parallel to that associated with DiGeorge/22q11.2 deletion syndrome (22q11.2DS). Disrupted coordination of tongue movement in LgDel mouse pups may contribute to suckling, feeding, and swallowing (S/F/S) disruptions that parallel pediatric dysphagia in infants and toddlers with 22q11.2DS. Using an in vitro rhythmically active medullary slice preparation, we found spontaneous firing as well as IPSC frequency differed significantly in neonatal LgDel versus wild-type (WT) protruder and retractor CNXII MNs that were identified by retrograde tracing from their target muscles. In response to respiration-related activity, initiation and decay of transiently increased firing in WT protruder MNs is delayed in LgDel, accompanied by altered excitatory/inhibitory (E/I) balance. In addition, LgDel retractor MNs have a transient increase in firing with diminished IPSC frequency that is not seen in WT. There were no significant differences in cell body volume of either XII class in WT and LgDel. Sholl analysis showed the total numbers of dendritic intersections (at 50- and 90-μm radii from the cell soma) were significantly greater for LgDel versus WT retractor MNs. Thus, the physiological, synaptic and cellular properties of distinct classes of CNXII MNs that coordinate tongue movement in neonatal WT mice are altered in LgDel. Such changes could contribute to sub-optimal coordination of S/F/S that underlies pediatric dysphagia in 22q11.2DS.

Development of electrophysiological and morphological properties of human embryonic stem cell-derived GABAergic interneurons at different times after transplantation into the mouse hippocampus.

Transplantation of human embryonic stem cell (hESC)-derived neural progenitors is a potential treatment for neurological disorders, but relatively little is known about the time course for human neuron maturation after transplantation and the emergence of morphological and electrophysiological properties. To address this gap, we transplanted hESC-derived human GABAergic interneuron progenitors into the mouse hippocampus, and then characterized their electrophysiological properties and dendritic arborizations after transplantation by means of ex vivo whole-cell patch clamp recording, followed by biocytin staining, confocal imaging and neuron reconstruction software. We asked whether particular electrophysiological and morphological properties showed maturation-dependent changes after transplantation. We also investigated whether the emergence of particular electrophysiological properties were linked to increased complexity of the dendritic arbors. Human neurons were classified into five distinct neuronal types (Type I-V), ranging from immature to mature fast-spiking interneurons. Hierarchical clustering of the dendritic morphology and Sholl analyses suggested four morphologically distinct classes (Class A-D), ranging from simple/immature to highly complex. Incorporating all of our data regardless of neuronal classification, we investigated whether any electrophysiological and morphological features correlated with time post-transplantation. This analysis demonstrated that both dendritic arbors and electrophysiological properties matured after transplantation.

Effects of 3D Stratification of Retinal Ganglion Cells in Sholl Analysis

BackgroundSholl analysis is used to quantify the dendritic complexity of neurons. Differences between two-dimensional (2D) and three-dimensional (3D) Sholl analysis can exist in neurons with extensive axial stratification of dendrites, however, in retinal ganglion cells (RGCs), only 2D analysis is typically reported despite varying degrees of stratification within the retinal inner plexiform layer. We determined the impact of this stratification by comparing 2D and 3D analysis of the same RGCs. New MethodTwelve retinas of mice expressing yellow fluorescent protein in RGCs under the control of the Thy1 promotor were whole-mounted. The entire dendritic arbor of 120 RGCs was traced, after which 2D and 3D Sholl analysis was performed. Two parameters describing dendritic complexity; area under the curve (AUC) and peak number of intersections (PNI) were then derived and analyzed. Results and Comparison with Existing MethodsThe AUC and PNI were significantly higher with 3D analysis compared to 2D analysis with medians of 2805 and 2508 units, and 31 and 27, respectively (P < 0.01). Both 2D and 3D AUC increased with arbor thickness. The discrepancy in AUC between the two methods depended on mean AUC (with every 1 unit increase in mean AUC resulting in a discrepancy of 0.1 unit), but not arbor thickness. ConclusionIn RGCs imaged in vitro, there is a difference in AUC and PNI derived with 2D and 3D Sholl analysis. Where possible, 3D Sholl analysis of RGCs should be performed for more accurate quantitative analysis of dendritic structure.

An automated data extraction and classification pipeline to identify a novel type of neuron within the dorsal striatum based on single-cell patch clamp and confocal imaging data.

We employed electrophysiological and fluorescence imaging techniques to describe the characteristics of a novel type of neuron discovered in the mouse dorsal striatum. Transgenic mice that express YFP-tagged channelrhodopsin-2 (ChR2) in neurons driven by the promoter for tyrosine hydroxylase (TH) were used and the intrinsic electrical properties of YFP-positive neurons in the dorsal striatum of these mice were characterized using whole-cell patch clamping in acute brain slices. Passive membrane properties - such as membrane capacitance, resting membrane potential and input resistance -and action potential properties- such as amplitude, kinetics and adaptation - were extracted from raw data files. Filling these neurons with neurobiotin enabled visualization of neuronal morphology via immunohistochemical labeling with streptavidin-conjugated fluorophore. Subsequent two-photon imaging allowed analyses of morphological properties such as somaticsize, dendritic branching (Sholl analysis) and density of dendritic spines. Unbiased analyses and hierarchical clustering of both morphological and functional data allowed us to identify a previously undescribed type of striatal neuron with unique properties. To facilitate identification of this new cell type, an end-to-end automated electrophysiology pipeline was developed that extracts relevant parameters and determines striatal neuron identity using neural-network based classifiers. These data and the software tool will permit other investigators to identify this novel type of neuron in their studiesand thereby better understand theroles thatthese neuronsplay in dorsal striatum circuitry.

Oligodendrocyte Response to Pathophysiological Conditions Triggered by Episode of Perinatal Hypoxia-Ischemia: Role of IGF-1 Secretion by Glial Cells

Differentiation of oligodendrocyte progenitors towards myelinating cells is influenced by a plethora of exogenous instructive signals. Insulin-like growth factor 1 (IGF-1) is one of the major factors regulating cell survival, proliferation, and maturation. Recently, there is an ever growing recognition concerning the role of autocrine/paracrine IGF-1 signaling in brain development and metabolism. Since oligodendrocyte functioning is altered after the neonatal hypoxic-ischemic (HI) insult, a question arises if the injury exerts any influence on the IGF-1 secreted by neural cells and how possibly the change in IGF-1 concentration affects oligodendrocyte growth. To quantify the secretory activity of neonatal glial cells, the step-wise approach by sequentially using the in vivo, ex vivo, and in vitro models of perinatal asphyxia was applied. A comparison of the results of in vivo and ex vivo studies allowed evaluating the role of autocrine/paracrine IGF-1 signaling. Accordingly, astroglia were indicated to be the main local source of IGF-1 in the developing brain, and the factor secretion was shown to be significantly upregulated during the first 24 h after the hypoxic-ischemic insult. And conversely, the IGF-1 amounts released by oligodendrocytes and microglia significantly decreased. A morphometric examination of oligodendrocyte differentiation by means of the Sholl analysis showed that the treatment with low IGF-1 doses markedly improved the branching of oligodendroglial cell processes and, in this way, promoted their differentiation. The changes in the IGF-1 amounts in the nervous tissue after HI might contribute to the resulting white matter disorders, observed in newborn children who experienced perinatal asphyxia. Pharmacological modulation of IGF-1 secretion by neural cells could be reasonable solution in studies aimed at searching for therapies alleviating the consequences of perinatal asphyxia.

New morphological features based on the Sholl analysis for automatic classification of traced neurons

BackgroundThis article addresses the automatic classification of reconstructed neurons through their morphological features. The purpose was to extend the capabilities of the L-Measure software. MethodsNew morphological features were developed, based on modifications of the conventional Sholl analysis. The lengths of the compartments, as well as their volumes, were added to the features used in the classical analysis in order to improve the results during automatic neuron classification. FSM were used to obtain subsets of lower cardinality from the full feature sets and the usefulness of these subsets was tested through their use in supervised classification tasks. The study was based on two types of neurons belonging to mice: pyramidal and GABAergic interneurons. Furthermore, a set of pyramidal neurons belonging to Later 4 and Layer 5 was analyzed. ResultsRF classifier shown the best performance combined with a Wrapper method.U-WNAD set allowed to obtain higher values than WN, A and D in all cases and better results than LM for the filters and wrappers FSM. U-LM-WNAD set, led to the highest AUC values for all the FSM studied. Similar results for different regions of cortex were obtained. Comparison with Existing Methods The new features exhibited high discriminatory power with which the values of AUC and Acc obtained in the experiments exceeded those obtained using only the features provided by L-Measure. ConclusionsThe highest values of AUC and Acc were obtained from the sets U-WNAD and U-LM-WNAD, evidencing the discriminatory power of the new proposed features.

Iron Deficiency and Iron Excess Alter Dendritic Architecture of Pyramidal Neurons in the Hippocampus of Neonatal Pigs

Abstract Objectives The study assessed effect of dietary iron on iron homeostasis and dendritic architecture of hippocampal neurons in young piglets. Methods On postnatal day (PD) 1, 30 piglets (15 male/female) were blocked by sex and randomized to treatments by receiving no (N), low (L) or high (H) dose of iron supplement during pre- (PD1–21) and post-weaning period (PD22–35). Pigs in N, L, and H group orally received 0, 1, and 30 mg iron/(kg BW· d) as ferrous sulfate solution pre-weaning and were fed a solid diet containing 30, 125, and 1000 mg iron/kg post-weaning, respectively. Blood samples were collected on PD1 and weekly thereafter to analyze iron biomarkers. Iron homeostasis in hippocampus was assessed by measuring gene and protein expression of iron transporters. Hippocampal neurons stained with Golgi-Cox method were traced and 3D reconstructed using Neurolucida. Dendritic arborization were quantified through Sholl analysis and Neurolucida Explorer. Results Pigs in H group had the highest growth rate, whereas N pigs displayed growth retardation from PD27 to PD35, resulting in significant difference in body weight compared to H group (P &amp;lt; 0.05). Iron dose-dependently increased hemoglobin (Hb), hematocrit, plasma iron and transferrin saturation since PD7 or PD14 (P &amp;lt; 0.05). Pigs in N group became iron deficient since PD14 (Hb &amp;lt; 11 g/dL). Similarly, hippocampal ferritin expression was upregulated with the increase of iron (P &amp;lt; 0.05). The mRNA expression of TFRC and DMT1 in hippocampus was highest in N and lowest in H group (P &amp;lt; 0.05). Despite relatively low abundance, HAMP expression tended to be higher in H than that in N (P &amp;lt; 0.10). Sholl analysis uncovered significant main effect of treatment on basilar dendritic arborization of CA1 and CA3 pyramidal neurons (P ≤ 0.04). There were less branching nodes and dendrites in N than in H group (P &amp;lt; 0.05). However, the difference in dendritic arborization was primarily derived from the higher order (&amp;gt; 3) of branches in both regions. Iron supplementation did not affect architecture of apical dendrites of granule cells in dentate gyrus. Conclusions Early-life iron status affects hippocampal iron homeostasis and alters development of pyramidal neurons in a piglet model. Funding Sources NIFA Hatch/Multistate Research Fund.

Automated high content image analysis of dendritic arborization in primary mouse hippocampal and rat cortical neurons in culture

BackgroundPrimary neuronal cell cultures are useful for studying mechanisms that influence dendritic morphology during normal development and in response to various stressors. However, analyzing dendritic morphology is challenging, particularly in cultures with high cell density, and manual methods of selecting neurons and tracing dendritic arbors can introduce significant bias, and are labor-intensive. To overcome these challenges, semi-automated and automated methods are being developed, with most software solutions requiring computer-assisted dendrite tracing with subsequent quantification of various parameters of dendritic morphology, such as Sholl analysis. However fully automated approaches for classic Sholl analysis of dendritic complexity are not currently available. New MethodThe previously described Omnisphero software, was extended by adding new functions to automatically assess dendritic mass, total length of the dendritic arbor and the number of primary dendrites, branch points, and terminal tips, and to perform Sholl analysis. ResultsThe new functions for assessing dendritic morphology were validated using primary mouse hippocampal and rat cortical neurons transfected with a fluorescently tagged MAP2 cDNA construct. These functions allow users to select specific populations of neurons as a training set for subsequent automated selection of labeled neurons in high-density cultures. Comparison with Existing Semi-Automated MethodsCompared to manual or semi-automated analyses of dendritic arborization, the new functions increase throughput while significantly decreasing researcher bias associated with neuron selection, tracing, and thresholding. ConclusionThese results demonstrate the importance of using unbiased automated methods to mitigate experimenter-dependent bias in analyzing dendritic morphology.

Morphological multivariate cluster analysis of murine retinal ganglion cells selectively expressing yellow fluorescent protein

Optic neuropathies, such as glaucoma, lead to retinal ganglion cell (RGC) death. Transgenic mouse strains that express fluorescent proteins under the control of the Thy1 promoter have permitted single RGC imaging. Specifically, in one strain of mice expressing yellow fluorescent protein (Thy1-YFP), fluorescence is expressed in only 0.2% of RGCs. This reduced expression allows visualization of the full dendritic arbour of YFP-expressing RGCs, facilitating the investigation of structural changes. As susceptibility amongst RGCs varies with morphology and subtype, labelling methods should ideally non-discriminately label RGCs to accurately determine the effects of experimental glaucoma. This study therefore sought to determine morphological subtypes of RGCs in the Thy1-YFP mouse strain. Retinas from Thy1-YFP mice were imaged ex vivo with fluorescence microscopy. With Sholl analysis, a technique for quantifying the morphology of individual neurons, the dendritic field (DF), area under the curve (AUC), normalized AUC (Nav), peak number of intersections (PNI), and skew for single RGCs were computed. The distance of the RGC from the optic nerve head (dONH) was also measured. These morphological parameters were inputted into a multivariate cluster analysis to determine the optimal number of clusters to group all RGCs analyzed, which were then grouped into “Small”, “Medium”, and “Large” sized cluster groups according to increasing DF size. A total of 178 RGCs from 10 retinas of 8 mice were analyzed from which the cluster analysis identified 13 clusters. Eighty-eight (49%), 77 (43.2%), and 13 (7.3%) RGCs were grouped into small, medium and large clusters, respectively. Clusters 1–6 had small DFs. Clusters 1 and 3 had the lowest AUC and Nav. Clusters 2, 3, and 5 had asymmetric DFs while Clusters 3, 5, and 6 were distal to the ONH. Clusters 7–11 had medium DFs; of these, Clusters 7 and 10 had the lowest AUC, Clusters 8 and 10 had the highest skew, and Clusters 7 and 11 were closest to the ONH. Clusters 12 and 13 had large DFs. Both had low skew and high AUC. High PNI and dONH distinguished Cluster 12 from Cluster 13. We present the largest study to date examining YFP expression in RGCs of transgenic Thy1-YFP mice. Among the 13 clusters, there was a wide range of morphological features with further variation within size categories. Our findings support the notion that YFP is expressed non-discriminatingly in RGCs of Thy1-YFP transgenic mice and this strain is a valuable tool for studies of experimental optic neuropathies.

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

Microglia Activation and IL‐1β Expression in Select Ventilatory Control Regions Following Exposure to Chronic Sustained Hypoxia

Ventilatory acclimatization to hypoxia (VAH) is defined as the time‐dependent increase in ventilation which occurs with chronic sustained hypoxia (CSH) of several hours to months. Previous research has shown that astrocytes and microglia undergo a morphology shift, indicating a possible change in activity, upon exposure to hypoxic conditions and may contribute to VAH. Understanding when and how the different cell types in respiratory control regions are activated is pertinent to understanding ventilatory control during hypoxic conditions. The first part of this study aimed to optimize the Sholl analysis method to detect morphological changes in microglia. Two different microglia antibodies, Iba‐1 and CD11b[Ox42], were compared using Sholl analysis to determine which antibody best represents the branching pattern of the individual microglia. Analysis indicated that neither antibody was statistically different from the other in terms of microglia branching (p&gt;0.05), so CD11b[Ox42] was chosen to simplify the immunohistochemistry protocol. The second part of this study assessed the morphology shift of microglia in the nucleus tractus solitarius (NTS) and Pre‐Bötzinger Complex (PBC) following CSH exposure. Based on previous research, we hypothesized that microglia in the NTS and PBC would be activated following CSH exposure, as assessed via a morphology shift to a more amoeboid state indicated by less microglia branch crossings of the Sholl brackets. To address this hypothesis, rats were exposed to either normoxic, 60‐minutes of CSH, or 12‐hours of CSH. Microglia morphology was assessed in perfused brainstem tissue via immunohistochemistry, confocal imaging, and image analysis. In the NTS, microglia branching analysis revealed a trend towards a more amoeboid morphology at the 60‐minute CSH time point, but only one of the Sholl analysis brackets, 21–30μM, was statistically significant from normoxic conditions (p&lt;0.05). In the PBC, microglia branching analysis also revealed a trend towards a more amoeboid morphology at the 60‐minute CSH time point with statistically significant (p&lt;0.05) branch patterns at the 11–20μM, 21–30μM, and 31–40 μM Sholl brackets. A morphology shift of microglia to a more amoeboid state could indicate their localized response to neurotransmitters or cytokines. In a follow‐up study we investigated the expression of IL‐1β in the NTS region using immunofluorescent labeling. Rat brainstem tissue from normoxic conditions, 15‐minute CSH, and 60‐minute CSH was labeled with antibodies against IL‐1β, GFAP, and Cd11b[Ox42]. IL‐1β positive cells were counted in the NTS region. This preliminary set of cell counts (n=2) suggests that there is an increase in IL‐1β positive cells with CHS exposure (normoxic 17 ± 2 IL‐1β positive cells, 15‐minute CSH 31.5 ± 0.5 IL‐1β positive cells, 60‐minute CSH 29.5 ± 9.5 IL‐1β positive cells). Taken together these data provide a great starting point in assessing the activation profiles of glial cells and cytokine activity in select respiratory control regions in the brainstem.Support or Funding InformationCentenary College Student‐Faculty Summer Research Award 2018 and 2019 (JRF and JAS); NIH R01HL081823 (FLP)

Vagotomy Alters Astrocyte Morphology in the Brainstem Nucleus Tractus Solitarii

The nucleus tractus solitarii (nTS) in the brainstem is the initial site of integration and modulation of sensory input from the cardiorespiratory system. The nTS is critical for maintaining blood pressure (BP), heart rate (HR), and respiratory homeostasis, and is an essential part of the reflex response to hypoxia. Alterations in both afferent input and nTS function occur in many cardiorespiratory conditions, including obstructive sleep apnea, hypertension, and heart failure. Visceral sensory information is provided to the nTS, in part, by afferents from the vagus nerves. Vagal afferents activate both neurons and astrocytes to modify nTS output and cardiorespiratory function. Vagotomy has been shown to decrease nTS neuronal activity, but whether this is a direct effect on neurons or indirect due to effects on astrocytes is unknown. We hypothesized that decreased afferent input (vagotomy) modifies nTS astrocyte morphology, and is associated with impaired cardiorespiratory responses to hypoxia. To test this hypothesis, male Sprague‐Dawley rats (5–7 weeks old) underwent either right cervical vagotomy caudal to the nodose (n=8), sham surgery (n=7), or no surgery/control (n=4). Prior to surgery and one week post‐surgery, cardiorespiratory parameters (respiration, oxygen saturation, HR, BP) were measured in conscious and anesthetized animals under baseline and hypoxic conditions (8–12% O2). Rats were euthanized and brains were examined across the caudal‐to‐rostral nTS. Immunohistochemistry was used to evaluate astrocytes with glial fibrillary acidic protein (GFAP) and S100 calcium‐binding protein B (S100B), and glutamatergic synapses with vesicular glutamate transporter 2 (VGLUT2) in the nTS. Immunoreactivity (‐IR) was compared between the left (intact) and right (manipulated) nTS among groups. Vagotomy increased astrocytic GFAP‐IR compared to sham and controls in both the intact and manipulated sides, primarily at the level of the area postrema. Sholl analysis of astrocyte processes showed that vagotomy increased the number of astrocyte branch segments and branchpoints compared to controls on the manipulated side, and both vagotomy and sham increased branching on the manipulated side compared to controls. The latter occurred at unique proximal and distal distances from the cell body. The number of S100B‐IR astrocytes increased in vagotomy (compared to sham) in the intact side, with a trend in the manipulated side. VGLUT2‐IR was not different among groups or between sides, suggesting a similar number of glutamate synapses and lack of their pruning with vagotomy. Cardiorespiratory responses to hypoxia were not altered after vagotomy. These data suggest decreased afferent input increases astrocyte number and morphology, which is likely due to reduced afferent activity rather than presynaptic afferent structural alterations. Thus, afferent input dynamically influences astrocyte structure and likely function to modify neuronal activity, further elucidating the importance of nTS astrocytes in cardiorespiratory homeostasis and disease.Support or Funding InformationT32 OD011126, R01 HL132836, R01 HL128454

Neuroinflammation and Age‐Dependent Salt‐Sensitive Hypertension

AimNormal aging and elevated dietary sodium intake have both been identified as contributors to hypertension (HTN). Further, recent data from multiple animal models suggest a role of central inflammation in HTN. Therefore, we speculate that central inflammation plays a role in age‐related salt‐sensitive HTN.MethodsMale Sprague‐Dawley rats aged 3, 8, and 16 months (mo) old were randomly assigned to a normal salt (NS; 0.6% NaCl) or high salt (HS; 4% NaCl) diet for 21 days (n=4–6/group). In a subset of animals (n=6/group) MAP was assessed via femoral artery cannulation. Sympathetic tone was assessed via renal norepinephrine (NE) measured by ELISA on day 21 in a subset of animals (n=6/group). Additional groups of rats were sacrificed via transcardial perfusion using phosphate‐buffered saline and 4% paraformaldehyde (PFA). Brains were extracted and fixed in 4% PFA and 30% sucrose. Immunohistochemistry was performed for markers of microglial (CD11b/c) and astrocyte activation (GFAP) in the paraventricular nucleus (PVN) of the hypothalamus, and assessed using ImageJ software.ResultsWhen fed a NS diet, male rats exhibit age‐dependent HTN (MAP [mmHg] 3 mo 124 ± 2 vs. 8 mo 135 ± 4 vs 16 mo 149 ± 3, p&lt;0.05). When fed a 21‐day HS diet, male rats develop salt‐sensitivite HTN (MAP [mmHg] 3 mo 126 ± 3 vs. 8 mo 143 ± 5 vs. 16 mo 169 ± 1, p&lt;0.5). Aged rats display elevated levels of renal NE, with impaired HS‐evoked suppression of sympathetic tone (renal NE content [pg/mg] 3 mo NS 612 ± 36 vs HS 368 ± 32; 8 mo NS 835 ± 48 vs HS 722 ± 44, p&lt;0.05; 16 mo HS 974 ± 13 vs NS 1019 ± 34, ns). There was no statistical difference between branching complexities on trend lines from Sholl analysis of microglia in all groups. On a NS diet, there is no change in astrocytic density in the PVN with age, (total astrocyte % tissue density: 3 mo 41.58 ± 6.00% vs. 8 mo 43.58 ± 3.65% vs. 16 mo 47.18 ± 3.76%, ns). In 3 mo and 8 mo rats, which do not get salt‐sensitive HTN, there is a significant increase in the activation of astrocytes in the PVN when fed a high salt diet (total astrocyte % tissue density: 3 mo NS 41.58 ± 6.00% vs. 3 mo HS 56.66 ± 4.51%, p&lt;0.05; 8 mo NS 43.58 ± 3.65% vs. 8 mo HS 58.28 ± 2.63%, p&lt;0.05). Sixteen month old rats, which become salt‐sensitive and hypertensive with age, exhibit a failure to increase PVN astrocyte levels on a high salt diet (total astrocyte % tissue density: NS 47.18 ± 3.76% vs. HS 50.66 ± 5.01%).ConclusionsMale Sprague‐Dawley rats become hypertensive and salt‐sensitive with age. On a NS diet, inflammation does not play a role in age‐dependent HTN. However, inflammation plays a role in the salt‐sensitivity of blood pressure mediated by astrocyte reactivity. Our findings propose that with age, PVN astrocytes express a loss of a protective mechanism when challenged with a high salt diet.Support or Funding InformationR01 AG062515, R01 HL139867, R01 HL141406, and R56 AG057687 to RDW

Differentiation and characterization of neurons derived from rat iPSCs

BackgroundInduced pluripotent stem cells (iPSCs) may be an advantageous source of neuronal cells to repair damage due to neurological disorders or trauma. Additionally, they are promising candidates to develop models to study underlying mechanisms of neurodegenerative diseases. While successful neural differentiation of iPSCs has been reported in mice, protocols detailing the generation of neural cells from rat iPSCs are relatively limited, and their optimization by manipulating cell culture methods has remained unexplored. New methodHere, we describe and compare the effects of four distinct, commonly used substrates on the neuronal differentiation of rat iPSC (riPSC) derived-neural progenitor cells. Our approach is to use substrate coating as a method to enrich differentiated riPSCs for neuronal subtypes with the desired morphology and maturity. We use a combination of electrophysiology, immunofluorescence staining, and Sholl analysis to characterize the cells generated on each substrate over a nine-day time course. ResultsThe surface coating presented by the cell culture substrate influences the polarity and arborization of differentiating neurons. Polyornithine-laminin coating promoted neuronal arborization and maturation, while Geltrex favored bipolar cells which displayed indicators of functional immaturity. Poly-d-lysine substrate was associated with limited neurite outgrowth and arborization. Gelatin was the least favorable substrate for the growth and differentiation of our cells.Comparison with Existing Method: Rat-derived neural progenitor cells have been previously derived; however, our methods to use substrate coatings to influence morphological and electrical maturity have not been explored previously. ConclusionSubstrate coatings can be selected to enrich differentiated riPSCs for distinctive neuronal morphologies.

Protein phosphatase PP2A regulates microtubule orientation and dendrite pruning in Drosophila.

Pruning that selectively eliminates inappropriate projections is crucial for sculpting neural circuits during development. During Drosophila metamorphosis, ddaC sensory neurons undergo dendrite-specific pruning in response to the steroid hormone ecdysone. However, the understanding of the molecular mechanisms underlying dendrite pruning remains incomplete. Here, we show that protein phosphatase 2A (PP2A) is required for dendrite pruning. The catalytic (Microtubule star/Mts), scaffolding (PP2A-29B), and two regulatory subunits (Widerborst/Wdb and Twins/Tws) play important roles in dendrite pruning. Functional analyses indicate that PP2A, via Wdb, facilitates the expression of Sox14 and Mical prior to dendrite pruning. Furthermore, PP2A, via Tws, governs the minus-end-out orientation of microtubules (MTs) in the dendrites. Moreover, the levels of Klp10A, a MT depolymerase, increase when PP2A is compromised. Attenuation of Klp10A fully rescues the MT orientation defects in mts or pp2a-29b RNAi ddaC neurons, suggesting that PP2A governs dendritic MT orientation by suppressing Klp10A levels and/or function. Taken together, this study sheds light on a novel function of PP2A in regulating dendrite pruning and dendritic MT polarity in sensory neurons.

Evaluation of Phytosomal Curcumin as an Anti-inflammatory Agent for Chronic Glial Activation in the GFAP-IL6 Mouse Model.

Chronic glial activation is characterized by an increased number of activated microglia and astroglia; these secrete free radicals and cytotoxic cytokines, subsequently causing neuronal damage. This study investigated the hypothesis that a soy-lecithin based phytosomal curcumin formulation can decrease glial activation in the brains of GFAP-IL6 mice, a model of chronic glial activation, which exhibits gliosis in various regions of the brain. Three doses of Meriva curcumin (MC) (874, 436, and 218 PPM) were fed to 3-month-old GFAP-IL6 and wild-type (WT) mice for 4 weeks. As markers of glial activation, the total numbers of Iba-1+ and TSPO+ microglia and macrophages, and GFAP+ astrocytes, were determined in the cerebellum and hippocampus by immunohistochemistry and unbiased stereology. Furthermore, the morphology of the glial cells was assessed by confocal microscopy and Sholl analysis. Administration of phytosomal curcumin led to a dose-dependent reduction in neuroinflammatory markers. Phytosomal curcumin (874 PPM) decreased the number of microglia by 26.2% in the hippocampus and by 48% in the cerebellum of the GFAP-IL6 mice compared with the GFAP-IL6 mice on normal food. Additionally, GFAP+ astrocyte numbers in the hippocampus of the GFAP-IL6 mice were decreased by 42%. The GFAP-IL6 mice exhibited a different microglial morphology to the WT mice, showing an increased soma size and perimeter. This difference was significantly reduced by the 874 PPM phytosomal curcumin dose. Our findings demonstrate that phytosomal curcumin is able to attenuate the inflammatory pathology, and potentially reverse the detrimental effects of chronic glial activation.

Oxycodone Decreases Dendritic Complexity in Female but not Male Rat Striatal Neurons In Vitro

The use of oxycodone in the past two decades has dramatically risen, yet the amount of research regarding how it impacts neuronal health is lacking. As prescription use and misuse in women of reproductive age increases there has been a corresponding increase in the number of infants who have been exposed to oxycodone in utero. Given the critical role of the striatum in motor control and reward regulation, the aim of the current study was to examine the effects of oxycodone on developing rat striatal neurons. Sex-specific effects of oxycodone on neuronal cytoarchitecture were examined in cultured rat striatal neurons with a primary focus on dendritic arborization. Neurons were extracted from either male or female embryonic day 18 rat striata and cultured and exposed to varying concentrations of oxycodone over a ten-day period. Dendritic complexity of the neurons was measured using Sholl analysis. Results indicate that oxycodone inhibits dendritic complexity in a dose-dependent manner in female but not male striatal neurons. Additional analysis indicated the number of non-primary dendrites in female striatal neurons significantly decreased with increasing concentrations of oxycodone, while the number of primary dendrites as well as the length of primary and non-primary dendrites was unaffected by oxycodone treatment in both sexes. These in vitro findings demonstrate sex-specific effects of oxycodone on the development of striatal dendritic architecture which may be important for understanding the effects of oxycodone exposure in utero.

Microtubule Minus-End Binding Protein CAMSAP2 and Kinesin-14 Motor KIFC3 Control Dendritic Microtubule Organization

SummaryNeuronal dendrites are characterized by an anti-parallel microtubule organization. The mixed oriented microtubules promote dendrite development and facilitate polarized cargo trafficking; however, the mechanism that regulates dendritic microtubule organization is still unclear. Here, we found that the kinesin-14 motor KIFC3 is important for organizing dendritic microtubules and to control dendrite development. The kinesin-14 motor proteins (Drosophila melanogaster Ncd, Saccharomyces cerevisiae Kar3, Saccharomyces pombe Pkl1, and Xenopus laevis XCTK2) are characterized by a C-terminal motor domain and are well described to organize the spindle microtubule during mitosis using an additional microtubule binding site in the N terminus [1, 2, 3, 4]. In mammals, there are three kinesin-14 members, KIFC1, KIFC2, and KIFC3. It was recently shown that KIFC1 is important for organizing axonal microtubules in neurons, a process that depends on the two microtubule-interacting domains [5]. Unlike KIFC1, KIFC2 and KIFC3 lack the N-terminal microtubule binding domain and only have one microtubule-interacting domain, the motor domain [6, 7]. Thus, in order to regulate microtubule-microtubule crosslinking or sliding, KIFC2 and KIFC3 need to interact with additional microtubule binding proteins to connect two microtubules. We found that KIFC3 has a dendrite-specific distribution and interacts with microtubule minus-end binding protein CAMSAP2. Depletion of KIFC3 or CAMSAP2 results in increased microtubule dynamics during dendritic development. We propose a model in which CAMSAP2 anchors KIFC3 at microtubule minus ends and immobilizes microtubule arrays in dendrites.

Dendritic complexity change in the triple transgenic mouse model of Alzheimer's disease.

Alzheimer’s disease (AD) is an irreversible, neurodegenerative disease that is characterized by memory impairment and executive dysfunction. However, the change of fine structure of neuronal morphology remains unclear in the AD model mouse. In this study, high-resolution mouse brain sectional images were scanned by Micro-Optical Sectioning Tomography (MOST) technology and reconstructed three-dimensionally to obtain the pyramidal neurons. The method of Sholl analysis was performed to analyze the neurons in the brains of 6- and 12-month-old AD mice. The results showed that dendritic complexity was not affected in the entorhinal cortex between 6-month-old mice and 12-month-old mice. The dendritic complexity had increased in the primary motor cortex and CA1 region of hippocampus of 12- month-old mice compared with 6-month-old mice. On the contrary, dendritic complexity in the prefrontal cortex was decreased significantly between 6-month-old and 12-month-old mice. To our knowledge, this is the first study to provide high-resolution brain images of triple transgenic AD mice for statistically analyzing neuronal dendrite complexity by MOST technology to reveal the morphological changes of neurons during AD progression.