Number Of Primary Dendrites Research Articles

Overexpression of α-Klotho isoforms promotes distinct Effects on BDNF-Induced Alterations in Dendritic Morphology

α-Klotho (α-Kl) is a modulator of aging, neuroprotection, and cognition. Transcription of the Klotho gene produces two splice variants—a membrane protein (mKl), which can be cleaved and released into the extracellular milieu, and a truncated secreted form (sKl). Despite mounting evidence supporting a role for α-Kl in brain function, the specific roles of α-Kl isoforms in neuronal development remain elusive. Here, we examined α-Kl protein levels in rat brain and observed region-specific expression in the adult that differs between isoforms. In the developing hippocampus, levels of isoforms decrease after the third postnatal week, marking the end of the critical period for development. We overexpressed α-Kl isoforms in primary cultures of rat cortical neurons and evaluated effects on brain-derived neurotrophic factor (BDNF) signaling. Overexpression of either isoform attenuated BDNF-mediated signaling and reduced intracellular Ca2+ levels, with mKl promoting a greater effect. mKl or sKl overexpression in hippocampal neurons resulted in a partially overlapping reduction in secondary dendrite branching. Moreover, mKl overexpression increased primary dendrite number. BDNF treatment of neurons overexpressing sKl resulted in a dendrite branching phenotype similar to control neurons. In neurons overexpressing mKl, BDNF treatment restored branching of secondary and higher order dendrites close, but not distal, to the soma. Taken together, the data presented support the idea that sKl and mKl play distinct roles in neuronal development, and specifically, in dendrite morphogenesis.

Tanshinone IIA Regulates Synaptic Plasticity in Mg2+-Free-Induced Epileptic Hippocampal Neurons via the PI3K/Akt Signaling Pathway.

Tanshinone IIA (TSIIA) is an element of the effective ingredients of Salvia miltiorrhiza Bunge (Labiatae), exhibits a significant therapeutic effect in brain neuroprotection. The focus of this study was the examination of synaptic plasticity of in Mg2+-free-induced epileptic hippocampus neurons and how TSIIA protects against it. The purity of the primary hippocampal neurons extracted from Sprague Dawley rats was assessed within 24 hours by microtubule-associated protein (MAP2) immunofluorescence staining. A hippocampal neuron model for Mg2+-free-induced spontaneous recurrent epileptiform discharge was developed, five experimental groups were then randomized: blank (Blank), model (Model), TSIIA (TSIIA, 20 µM), LY294002 (LY294002, 25 µM), and TSIIA+LY294002 (TSIIA+LY294002, 20 µM+25 µM). FIJI software was used to examine variations of neurite complexity, total length of hippocampal neurons, number of primary dendrites and density of dendritic spines. Developmental regulation brain protein (Drebrin) and brain-derived neurotrophic factor (BDNF) expression was evaluated using immunofluorescence staining and the relative expression of phospho-protein kinase B (p-Akt)/Akt, BDNF, synaptophysin (SYN) and postsynaptic density 95 (PSD-95) determined by Western blot. In contrast to the model group, TSIIA drastically reduced damage to synaptic plasticity of hippocampal neurons caused by epilepsy (p < 0.05). The TSIIA group showed a significant increase in the relative expression of PSD-95, SYN, BDNF, and p-Akt/Akt (p < 0.01). TSIIA was effective in reducing harm to the synaptic plasticity of hippocampal neurons induced by persistent status epilepticus, with the possible mechanism being regulation of the phosphatidylinositol 3-kinase 56 (PI3K)/Akt signaling pathway.

Development of Pleiotropic TrkB and 5-HT4 Receptor Ligands as Neuroprotective Agents.

One common event that is the most detrimental in neurodegenerative disorders, even though they have a complex pathogenesis, is the increased rate of neuronal death. Endogenous neurotrophins consist of the major neuroprotective factors, while brain-derived neurotrophic factor (BDNF) and its high-affinity tyrosine kinase receptor TrkB are described in a number of studies for their important neuronal effects. Normal function of this receptor is crucial for neuronal survival, differentiation, and synaptic function. However, studies have shown that besides direct activation, the TrkB receptor can be transactivated via GPCRs. It has been proven that activation of the 5-HT4 receptor and transactivation of the TrkB receptor have a positive influence on neuronal differentiation (total dendritic length, number of primary dendrites, and branching index). Because of that and based on the main structural characteristics of LM22A-4, a known activator of the TrkB receptor, and RS67333, a partial 5-HT4 receptor agonist, we have designed and synthesized a small data set of novel compounds with potential dual activities in order to not only prevent neuronal death, but also to induce neuronal differentiation in neurodegenerative disorders.

Brain-specific Pd1 deficiency leads to cortical neurogenesis defects and depressive-like behaviors in mice.

Embryonic neurogenesis is tightly regulated by multiple factors to ensure the precise development of the cortex. Deficiency in neurogenesis may result in behavioral abnormalities. Pd1 is a well-known inhibitory immune molecule, but its function in brain development remains unknown. Here, we find brain specific deletion of Pd1 results in abnormal cortical neurogenesis, including enhanced proliferation of neural progenitors and reduced neuronal differentiation. In addition, neurons in Pd1 knockout mice exhibit abnormal morphology, both the total length and the number of primary dendrites were reduced. Moreover, Pd1cKO mice exhibit depressive-like behaviors, including immobility, despair, and anhedonia. Mechanistically, Pd1 regulates embryonic neurogenesis by targeting Pax3 through the β-catenin signaling pathway. The constitutive expression of Pax3 partly rescues the deficiency of neurogenesis in the Pd1 deleted embryonic brain. Besides, the administration of β-catenin inhibitor, XAV939, not only rescues abnormal brain development but also ameliorates depressive-like behaviors in Pd1cKO mice. Simultaneously, Pd1 plays a similar role in human neural progenitor cells (hNPCs) proliferation and differentiation. Taken together, our findings reveal the critical role and regulatory mechanism of Pd1 in embryonic neurogenesis and behavioral modulation, which could contribute to understanding immune molecules in brain development.

Postnatal Inhibition of TrkB Kinase Reduces the Number of Phrenic Motor Neurons

During embryonic development, the number of phrenic motor neurons (PhMNs) declines precipitously during the final trimester and continues into the early postnatal period. When BDNF/TrkB signaling is absent in utero, animals do not survive after birth, most likely due to excessive loss of vital motor neurons (e.g., PhMNs). Far less is known about the early postnatal effect of BDNF/TrkB signaling. We have shown that BDNF/TrkB signaling is important in promoting PhMN neuroplasticity in mature rodents. Taken together, we hypothesized that inhibition of BDNF/TrkB signaling during early postnatal development results in fewer PhMNs. Chemo-genetic TrkBF616A mutant mice were used to induce inhibition of TrkB kinase activity in response to oral administration (via drinking water) of a phosphoprotein phosphatase 1 inhibitor derivative (1NMPP1). 1NMPP1-induced TrkB kinase inhibition was initiated at P0 and continued until P21 and compared to untreated control mice. At P28, PhMNs were retrogradely labeled via rhodamine phrenic nerve dip. One day later, mice were euthanized, perfused with 4% paraformaldehyde, and the cervical spinal cord was excised and processed for longitudinal cryosectioning (70 μm). Labeled PhMNs were imaged by 3D high resolution confocal microscopy. The number of PhMNs were counted. PhMN somal surface area and number of primary dendrites were also determined. 1NMPP1-induced inhibition of TrkB kinase resulted in 24% fewer PhMNs (P=0.01) and 21% more primary dendrites (P=0.0002) than control mice. The distribution of PhMN somal surface areas was not affected. We conclude that BDNF/TrkB signaling is essential in promoting survival of PhMNs in the early postnatal period. BDNF/TrkB signaling during postnatal development also plays a role in paring primary dendrites. While inhibition of TrkB kinase did not significantly alter the distribution of PhMN somal surface areas at P28, it is possible that further growth of PhMNs into maturity might have been altered as larger PhMNs emerge as a result of disproportionate growth beyond this age in mice. Supported by National Institutes of Health grants R01-AG044615 (GCS), R01-HL96750 (GCS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diverse Morphology of Sympathetic Neuron Subpopulations in the Stellate Ganglia

Sympathetic nerves innervate the heart and changes in sympathetic efferent activity can modulate cardiac function. The post‐ganglionic sympathetic neurons that innervate the heart are located primarily in the stellate ganglia (SG), which also contains neurons that project to brown adipose tissue (BAT), among other target tissues. We aim to examine morphological changes in SG neurons after myocardial infarction, and characterize the specificity of neural plasticity within the ganglion. To interpret morphological changes induced by cardiac injury, we must first fully understand the normal morphology of distinct groups of SG neurons. To that end, we examined SG neurons following injections of retrograde tracers into cardiac tissue and/or intrascapular brown adipose tissue (BAT) in adult male and female C57BL/6J mice (8 males, 2 females, 28‐32 weeks, 24‐39 g; The Jackson Laboratory). Using cholera toxin B (CTb) tracers conjugated to two different fluorophores (1 mg/mL in 0.01 M PBS; Alexa Fluor 488 or Alexa Fluor 555), we assessed the number, dispersion, pre‐synaptic cholinergic inputs and morphology of BAT‐ and cardiac‐projecting SG neurons. We also examined the quality of the CTb tracer detection at varying times after BAT injection (7, 14 and 28 days; n=2 mice per time point). BAT‐projecting SG neuron visualization was optimal 7 days post‐injection compared to 14 and 28 days, and could be visualized directly or with antibodies against CTb. BAT‐projecting SG neurons were located in both left and right SG, predominately ipsilateral to the injection site and were widely dispersed. In contrast, cardiac‐projecting SG neurons were more localized near the base of the inferior cardiac nerve. In BAT‐ and cardiac‐injected mice (n=4), we found that SG neurons were not dual‐labeled, supporting the notion that sympathetic innervation to target tissues is specific. We found cardiac‐projecting SG neuron somas have more volume (mm3) and cardiac‐projecting cells are less abundant within the SG than BAT‐projecting neurons with our tracer‐labeling paradigm. We also discovered a positive correlation between number of primary dendrites per neuron and soma volume in cardiac‐projecting SG neurons, though not in BAT‐projecting SG neurons. Additionally, in both SG subpopulations, the number of cholinergic inputs marked with vesicular acetylcholine transporter (VAChT) on the cell body is positively correlated to soma volume. Tract tracing distinct subpopulations of neurons will allow us to characterize morphological changes after cardiac injury in specific cell groups. This approach will allow insights into whether mechanisms of plasticity are cell specific or occur globally within the SG.

Distinct morphology of cardiac- and brown adipose tissue-projecting neurons in the stellate ganglia of mice.

Sympathetic neurons that innervate the heart are located primarily in the stellate ganglia (SG), which also contains neurons that project to brown adipose tissue (BAT). These studies were designed to examine the morphology of these two populations (cardiac‐ and BAT‐projecting) and their target connectivity. We examined SG neurons in C57BL/6J mice following injections of the retrograde tracer cholera toxin B (CTb) conjugated to Alexa Fluor 488 and Alexa Fluor 555, into cardiac tissue and intrascapular BAT. BAT‐projecting SG neurons were widely dispersed in SG, while cardiac‐projecting SG neurons were localized primarily near the inferior cardiac nerve base. SG neurons were not dual‐labeled, suggesting that sympathetic innervation is specific to the heart and BAT, supporting the idea of “labeled lines” of efferents. Morphologically, cardiac‐projecting SG somata had more volume and were less abundant than BAT‐projecting neurons using our tracer‐labeling paradigm. We found a positive correlation between the number of primary dendrites per neuron and soma volume in cardiac‐projecting SG neurons, though not in BAT‐projecting neurons. In both SG subpopulations, the number of cholinergic inputs marked with vesicular acetylcholine transporter (VAChT) puncta contacting the soma was positively correlated to soma volume, suggesting scaling of inputs across a range of neuronal sizes. In separate studies using dual tracing from left and right BAT, we found that BAT‐projecting SG neurons were located predominately ipsilateral to the injection, but a small subset of SG neurons project bilaterally to BAT. This tracing approach will allow the assessment of cell‐specific mechanisms of plasticity within subpopulations of SG neurons.

Postnatal Phrenic Motor Neuron and Diaphragm Maturation in the Mouse

In rats, there is a strong association between the expansion of phrenic motor neuron (PhMN) size (from ~21‐28 days postnatal), the emergence of mature diaphragm muscle (DIAm) myosin heavy chain (MyHC2X and MyHC2B) isoform expression, alongside the manifestation of mature morphological and functional properties. Despite mice models being de rigueur for developmental investigations, the postnatal development of mouse PhMN and DIAm properties have not been comprehensively characterized. Using wildtype C57 mice at ages P7, P14, P21, P28, and &gt;3 months (maturity) we evaluated PhMN and DIAm physiologic properties. PhMNs were labelled via rhodamine phrenic nerve dip, processed for longitudinal cryosectioning (70 µm) and prepared for confocal imaging and PhMN assessment. Ex vivo DIAm strips were used for functional assessments of maximum specific force (Po) and fatigue. DIAm strips were also fresh‐frozen, with 8 µm cross‐sections immuno‐labelled for MyHCslow, MyHC2A, MyHC2X and MyHC2X and processed for fluorescent assessment. The number of PhMNs remained stable throughout postnatal life (~195), while PhMN surface area increased steadily, ~10% from P14 to P21, P21 to P28, and P28 to maturity, with overall ~30% increase from P14 to maturity. The biggest changes in number of primary dendrites occurred between P14 and P28 and between P28 and maturity, 25% decrease in primary dendrites between each group. DIAm maximum Po increased in two phases, 3.5‐fold from P7 to P14 and remained similar until P21. The second large increase was ~3‐fold from P21 to P28 and remained steady to maturity. The fatiguability of the DIAm increased with age in two phases, with fatigability being similar from P7 and P14 and increasing by ~20% at P21. Following, fatigability of the DIAm was similar from P21 to P28 and increased by ~35% at maturity. These major PhMN and DIAm changes coincide with the steady growth of PhMN and the emergence of MyHC2X and MyHC2B expression in DIAm from P14 to P21 onwards. The maturation of orderly PhMN recruitment likely depends on an interplay between PhMN size properties and network inputs to a refined dendritic tree. We show that DIAm muscle fiber maturation in mice hinges on the expression of MyHC2X and MyHC2B isoforms that is associated with an ~10 fold increase in DIAm force‐generation capacity.

Morphological alteration in rat hippocampal neuronal dendrites following chronic binge prenatal alcohol exposure

Prenatal alcohol exposure (PAE) may result in Fetal Alcohol Spectrum Disorders (FASD). The hippocampus has been recognized as a vulnerable target to alcohol-induced developmental damage. However, the effect of prenatal exposure to alcohol on dendritic morphological adaptations throughout the hippocampal fields in the developing brain still remains largely unknown in the context of FASD. We hypothesized that chronic binge alcohol exposure during pregnancy alters dendrite arborization throughout the developing rat hippocampus. Pregnant Sprague-Dawley rats were assigned to either a pair-fed control (PF-Cont) or a binge alcohol (Alcohol) treatment group. Alcohol dams were acclimatized via a once-daily orogastric gavage of 4.5 g/kg alcohol from gestational day (GD) 5–10 and progressed to 6 g/kg alcohol from GD 11–21. Pair-fed dams similarly received isocaloric maltose dextrin. After parturition, all dams received an ad libitum diet and nursed their offspring until postnatal day (PND) 10 when the pup brains were collected for morphological analysis. PAE increased dendritic arborization and complexities of CA1, CA2/3, and DG neurons in the PND 10 rat hippocampus. The number of primary dendrites, total dendritic length, and number of dendritic branches were significantly increased following PAE, and Sholl analysis revealed significantly more intersections of the dendritic processes in PND 10 offspring following PAE compared with those in the PF-Cont group. We conclude that chronic binge PAE significantly alters hippocampal dendritic morphology in the developing hippocampus. We conjecture that this morphological alteration in postnatal rat hippocampal dendrites following chronic binge prenatal alcohol exposure may play a critical role in FASD neurobiological phenotypes.

Cortactin deacetylation by HDAC6 and SIRT2 regulates neuronal migration and dendrite morphogenesis during cerebral cortex development

sProper dendrite morphogenesis and neuronal migration are crucial for cerebral cortex development and neural circuit formation. In this study, we sought to determine if the histone deacetylase HDAC6 plays a role in dendrite development and neuronal migration of pyramidal neurons during cerebral cortex development. It was observed that knockdown of HDAC6 leads to defective dendrite morphogenesis and abnormal Golgi polarization in vitro, and the expression of wild type cortactin or deacetyl-mimetic cortactin 9KR rescued the defective phenotypes of the HDAC6 knockdown neurons. This suggests that HDAC6 promotes dendritic growth and Golgi polarization through cortactin deacetylation in vitro. We also demonstrated that ectopic expression of SIRT2, a cytoplasmic NAD+ − dependent deacetylase, suppresses the defects of HDAC6 knockdown neurons. These results indicate that HDAC6 and SIRT2 may be functionally redundant during dendrite development. Neurons transfected with both HDAC6 and SIRT2 shRNA or acetyl-mimetic cortactin 9KQ showed slow radial migration compared to the control cells during cerebral cortex development. Furthermore, a large portion of cortactin 9KQ-expressing pyramidal neurons at layer II/III in the cerebral cortex failed to form an apical dendrite toward the pial surface and had an increased number of primary dendrites, and the percentage of neurons with dendritic Golgi decreased in cortactin 9KQ-expressing cells, compared to control neurons. Taken together, this study suggests that HDAC6 and SIRT2 regulate neuronal migration and dendrite development through cortactin deacetylation in vivo.

Plasticity of thoracic interneurones rostral to a lateral spinal cord lesion

The morphology and projections of ventral horn interneurones in the segment above an ipsilateral thoracic lateral spinal cord lesion were studied in the cat by intracellular injections of Neurobiotin at 6 to 18 weeks post-lesion and compared with previously published control data from uninjured spinal cords. The cell axons ascended, descended or both, mostly contralaterally and mostly spared by the lesion. Unusual morphological dendritic features were seen in the lesion group, mostly growth-related, including complex dendritic appendages, twisted or multiple-branched terminal dendrites, commissural dendrites, apparently swollen proximal dendrites and rostrocaudal asymmetries. Significant quantitative differences included more dendritic spines in the lesion group (3.4×) and smaller soma areas in the lesion group (with similar numbers of primary dendrites and rostrocaudal dendritic spans). Immunoreactivity to microtubule associated protein 2a/b was detected in the proximal, but not distal, dendrites of cells in the lesion group, corresponding to an overall decrease in immunoreactivity in the ventral horns on the lesion side compared to the other. For axon collaterals, significant increases for the lesion group were seen in the number of collaterals in the first 4 mm of axon and in the area of ventral/intermediate horn occupied by terminals, including increased innervation of some regions, among which were the intermediolateral columns. This dendritic and axonal plasticity makes the interneuones candidates for a role in detour circuits but also for a maladaptive role in autonomic hyperreflexia.

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.

Neuroprotective Effects of Grape Seed Procyanidins on Ethanol-Induced Injury and Oxidative Stress in Rat Hippocampal Neurons.

Ethanol is a small molecule capable of interacting with numerous targets in the brain, the mechanisms of which are complex and still poorly understood. Studies have revealed that ethanol-induced hippocampal neuronal injury is associated with oxidative stress. Grape seed procyanidin (GSP) is a new type of antioxidant that is believed to scavenge free radicals and be anti-inflammatory. This study evaluated the ability and mechanism by which the GSP improves ethanol-induced hippocampal neuronal injury. Primary cultures of hippocampal neurons were exposed to ethanol (11, 33 and 66mM, 1, 4, 8, 12 and 24h) and the neuroprotective effects of GSP were assessed by evaluating the activity of superoxide dismutase (SOD), the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH) and cell morphology. Our results indicated that GSP prevented ethanol-induced neuronal injury by reducing the levels of MDA and LDH, while increasing the activity of SOD. In addition, GSP increased the number of primary dendrites and total dendritic length per cell. Together with previous findings, these results lend further support to the significance of developing GSP as a therapeutic tool for use in the treatment of alcohol use disorders.

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.

Role of serotonin 4 receptor in the growth of hippocampal neurons during the embryonic development in mice

Serotonin (5-HT) homeostasis is critical for the brain development which influences neurogenesis, neuronal migration, and circuit formation. Distinctive distribution patterns of serotonin receptors (5-HTRs) in the brain govern various physiological activities. Amongst the 5-HTRs, serotonin 4 receptor (5-HT4R) is widely expressed in embryonic forebrain and affects neuronal development, synaptogenesis, and behavior, but its specific role in brain development is still not completely understood. Therefore, in the present study, we addressed the roles of 5-HT4R in the growth of hippocampal neurons during the development of mice brain. We cultured hippocampal neurons of the mouse at embryonic day 18 and then treatment of 5-HT4R agonist RS67333 was employed. We found RS67333 significantly increased the axonal length, diameter and branching along with total dendritic length, number of primary dendrites and their branching. In addition, these effects were neutralized by the concomitant treatment of 5-HT4R antagonist GR125487, which confirmed the specific role of the 5-HT4R in the growth of axon and dendrites. Further, the treatment of RS67333 upregulated the mRNA expression of collapsin response mediator protein-2 (CRMP2) and non-phosphorylated CRMP2 (npCRMP2) together with neurotrophic factors (BDNF, NT-3, NGF) and TRK-A. Additionally, the current research findings reveal that the knockdown of CRMP2 inhibited RS67333-induced growth of the axons and dendrites, which indicates that CRMP2 is required for the 5-HT4R-mediated growth of the axons and dendrites. Overall, the findings of the present in vitro study enrich the understanding and provide insight roles of 5-HT4R in embryonic brain development by promoting the growth of hippocampal neurons.

BDNF Induced Translation of Limk1 in Developing Neurons Regulates Dendrite Growth by Fine-Tuning Cofilin1 Activity.

Dendritic growth and branching are highly regulated processes and are essential for establishing proper neuronal connectivity. There is a critical phase of early dendrite development when these are heavily regulated by external cues such as trophic factors. Brain-derived neurotrophic factor (BDNF) is a major trophic factor known to enhance dendrite growth in cortical neurons, but the molecular underpinnings of this response are not completely understood. We have identified that BDNF induced translational regulation is an important mechanism governing dendrite development in cultured rat cortical neurons. We show that BDNF treatment for 1 h in young neurons leads to translational up-regulation of an important actin regulatory protein LIM domain kinase 1 (Limk1), increasing its level locally in the dendrites. Limk1 is a member of serine/threonine (Ser/Thr) family kinases downstream of the Rho-GTPase pathway. BDNF induced increase in Limk1 levels leads to increased phosphorylation of its target protein cofilin1. We observed that these changes are maintained for long durations of up to 48 h and are mediating increase in number of primary dendrites and total dendrite length. Thus, we show that BDNF induced protein synthesis leads to fine-tuning of the actin cytoskeletal reassembly and thereby mediate dendrite development.

Alpha6-Containing Nicotinic Acetylcholine Receptors Mediate Nicotine-Induced Structural Plasticity in Mouse and Human iPSC-Derived Dopaminergic Neurons.

Midbrain dopamine (DA) neurons are considered a critical substrate for the reinforcing and sensitizing effects of nicotine and tobacco dependence. While the role of the α4 and β2 subunit containing nicotinic acetylcholine receptors (α4β2∗nAChRs) in mediating nicotine effects on DA release and DA neuron activity has been widely explored, less information is available on their role in the morphological adaptation of the DA system to nicotine, eventually leading to dysfunctional behaviors observed in nicotine dependence. In particular, no information is available on the role of α6∗nAChRs in nicotine-induced structural plasticity in rodents and no direct evidence exists regarding the occurrence of structural plasticity in human DA neurons exposed to nicotine. To approach this problem, we used two parallel in vitro systems, mouse primary DA neuron cultures from E12.5 embryos and human DA neurons differentiated from induced pluripotent stem cells (iPSCs) of healthy donors, identified using TH+ immunoreactivity. In both systems, nicotine 1–10 μM produced a dose-dependent increase of maximal dendrite length, number of primary dendrites, and soma size when measured after 3 days in culture. These effects were blocked by pretreatments with the α6∗nAChR antagonists α-conotoxin MII and α-conotoxin PIA, as well as by the α4β2nAChR antagonist dihydro-β-erythroidine (DHβE) in both mouse and human DA neurons. Nicotine was also ineffective when the primary DA neurons were obtained from null mutant mice for either the α6 subunit or both the α4 and α6 subunits of nAChR. When pregnant mice were exposed to nicotine from gestational day 15, structural plasticity was also observed in the midbrain DA neurons of postnatal day 1 offspring only in wild-type mice and not in both null mutant mice. This study confirmed the critical role of α4α6∗nAChRs in mediating nicotine-induced structural plasticity in both mouse and human DA neurons, supporting the translational relevance of neurons differentiated from human iPSCs for pharmacological studies.

A potential gliovascular mechanism for microglial activation: differential phenotypic switching of microglia by endothelium versus astrocytes

BackgroundActivation of microglia can result in phenotypic and functional diversity. However, the pathways that trigger different states of microglial activation remain to be fully understood. Here, we hypothesized that after injury, astrocytes and endothelium may contribute to a gliovascular switch for microglial activation.MethodsAstrocytes or cerebral endothelial cells were subjected to oxygen glucose deprivation, then conditioned media were transferred to microglia. The release of TNFα, IL-1β, IL-10, and IGF-1 was measured using ELISA. Surface markers of CD11b, CD45, CD86, and MHC class II were detected by flow cytometry. mRNA expression of iNOS, CD86, CD206, Arginase1, and transcription factors was measured using real-time PCR. Microglial function including migration and phagocytosis was assessed. Dendritogenesis was determined by counting the number of primary dendrites, secondary dendrites, and dendritic ends in the neurons exposed to either endothelial- or astrocyte-activated microglia.ResultsExposure to conditioned media from oxygen-glucose-deprived cerebral endothelial cells or oxygen-glucose-deprived astrocytes activated microglia into different forms. The endothelium converted ramified microglia into amoeboid shapes; increased the release of TNFα, IL-1β, and IL-10; decreased IGF-1; upregulated iNOS expression; and inhibited microglial migration and phagocytosis. In contrast, astrocytes increased microglial production of IGF-1, upregulated CD206 expression, and enhanced microglial phagocytosis. These opposing effects of the endothelium versus astrocyte crosstalk partly mirror potentially deleterious versus potentially beneficial microglial phenotypes. Consistent with this idea, endothelial-activated microglia were neurotoxic, whereas astrocyte-activated microglia did not affect neuronal viability but instead promoted neuronal dendritogenesis.ConclusionThese findings provide proof of concept that endothelial cells and astrocytes provide differing signals to microglia that influence their activation states and suggest that a gliovascular switch may be involved in the balance between beneficial versus deleterious microglial properties.

Effects of prenatal binge-like ethanol exposure and maternal stress on postnatal morphological development of hippocampal neurons in rats

BackgroundAlcohol is one of the most commonly used drugs of abuse negatively affecting human health and it is known as a potent teratogen responsible for fetal alcohol syndrome (FAS), which is characterized by cognitive deficits especially pronounced in juveniles but ameliorating in adults. Searching for the potential morphological correlates of these effects, in this study, we compared the course of developmental changes in the morphology of principal hippocampal neurons in fetal-alcohol (A group), intubated control (IC group), and intact control male rats (C group) over a protracted period of the first two postnatal months. MethodsEthanol was administered to the pregnant Wistar dams intragastrically, throughout gestation days (GD) 7–20, at a total dose of 6g/kg/day resulting in the mean blood alcohol concentration (BAC) of 246.6±40.9mg/dl. Ten morphometric parameters of Golgi-stained hippocampal neurons (pyramidal and granule) from CA1, CA3, and DG areas were examined at critical postnatal days (PD): at birth (PD1), at the end of the brain growth spurt period (PD10), in juveniles (PD30), and in young adults (PD60). ResultsDuring postnatal development, the temporal pattern of morphometric changes was shown to be region-dependent with most significant alterations observed between PD1-30 in the CA region and between PD10-30 in the DG region. It was also parameter-dependent with the soma size (except for CA3 pyramids), number of primary dendrites, dendrite diameter, dendritic tortuosity and the branch angle demonstrating little changes, while the total dendritic field area, dendritic length, number of dendritic bifurcations, and spine density being highly increased in all hippocampal regions during the first postnatal month. Moderate ethanol intoxication and the maternal intubation stress during gestation, showed similar, transient effects on the neuron development manifested as a smaller soma size in granule cells, reduced dendritic parameters and lower spine density in pyramidal neurons at PD1. Full recovery from these effects took place within the first 10 postnatal days. ConclusionsThis study showed regional and temporal differences in the development of different morphometric features of principal hippocampal neurons in intact subjects over a protracted 2-months postnatal period. It also demonstrated an overlap in the effects of a moderate fetal ethanol intoxication and a mild maternal stress produced by the intragastric intubation, a commonly used method of ethanol administration to the pregnant dams. Fast recovery from the adverse effects on the soma size, dendritic arborization and spines density observed at birth indicates towards the fetal ethanol/stress induced developmental retardation.

Alterations of striatal indirect pathway neurons precede motor deficits in two mouse models of Huntington's disease

Striatal neurons forming the indirect pathway (iSPNs) are particularly vulnerable in Huntington's disease (HD). In this study we set out to investigate morphological and physiological alterations of iSPNs in two mouse models of HD with relatively slow disease progression (long CAG repeat R6/2 and zQ175-KI). Both were crossed with a transgenic mouse line expressing eGFP in iSPNs. Using the open-field and rotarod tests, we first defined two time points in relation to the occurrence of motor deficits in each model. Then, we investigated electrophysiological and morphological properties of iSPNs at both ages. Both HD models exhibited increased iSPN excitability already before the onset of motor deficits, associated with a reduced number of primary dendrites and decreased function of Kir- and voltage-gated potassium channels. Alterations that specifically occurred at symptomatic ages included increased calcium release by back-propagating action potentials in proximal dendrites, due to enhanced engagement of intracellular calcium stores. Moreover, motorically impaired mice of both HD models showed a reduction in iSPN spine density and progressive formation of huntingtin (Htt) aggregates in the striatum.Our study therefore reports iSPN-specific alterations relative to the development of a motor phenotype in two different mouse models of HD. While some alterations occur early and are partly non-progressive, others potentially provide a pathophysiological marker of an overt disease state.