Soma Size Research Articles

Phenylbutyrate administration reduces changes in the cerebellar Purkinje cells population in PDC-deficient mice

In humans, pyruvate dehydrogenase complex (PDC) deficiency impairs brain energy metabolism by reducing the availability of the functional acetyl‑CoA pool. This "hypometabolic defect" results in congenital lactic acidosis and abnormalities of brain morphology and function, ranging from mild ataxia to profound psychomotor retardation. Our previous study showed reduction in total cell number and dendritic arbors in the cerebellar Purkinje cells in systemic PDC‑deficient mice. Phenylbutyrate has been shown to increase PDC activity in cultured fibroblasts from PDC‑deficient patients. Hence, we investigated the effects of postnatal (days 2‑35) phenylbutyrate administration on the cerebellar Purkinje cell population in PDC‑deficient female mice. Histological analyses of different regions of cerebellar cortex from the brain‑specific PDC‑deficient saline‑injected mice revealed statistically significant reduction in the Purkinje cell density and increased cell size of the individual Purkinje cell soma compared to control PDC‑normal, saline‑injected group. Administration of phenylbutyrate to control mice did not cause significant changes in the Purkinje cell density and cell size in the studied regions. In contrast, administration of phenylbutyrate variably lessened the ill effects of PDC deficiency on Purkinje cell populations in different areas of the cerebellum. Our results lend further support for the possible use of phenylbutyrate as a potential treatment for PDC deficiency.

Comparative neocortical neuromorphology in felids: African lion, African leopard, and cheetah

The present study examines cortical neuronal morphology in the African lion (Panthera leo leo), African leopard (Panthera pardus pardus), and cheetah (Acinonyx jubatus jubatus). Tissue samples were removed from prefrontal, primary motor, and primary visual cortices and investigated with a Golgi stain and computer-assisted morphometry to provide somatodendritic measures of 652 neurons. Although neurons in the African lion were insufficiently impregnated for accurate quantitative dendritic measurements, descriptions of neuronal morphologies were still possible. Qualitatively, the range of spiny and aspiny neurons across the three species was similar to those observed in other felids, with typical pyramidal neurons being the most prominent neuronal type. Quantitatively, somatodendritic measures of typical pyramidal neurons in the cheetah were generally larger than in the African leopard, despite similar brain sizes. A MARsplines analysis of dendritic measures correctly differentiated 87.4% of complete typical pyramidal neurons between the African leopard and cheetah. In addition, unbiased stereology was used to compare the soma size of typical pyramidal neurons (n = 2,238) across all three cortical regions and gigantopyramidal neurons (n = 1,189) in primary motor and primary visual cortices. Both morphological and stereological analyses indicated that primary motor gigantopyramidal neurons were exceptionally large across all three felids compared to other carnivores, possibly due to specializations related to the felid musculoskeletal systems. The large size of these neurons in the cheetah which, unlike lions and leopards, does not belong to the Panthera genus, suggests that exceptionally enlarged primary motor gigantopyramidal neurons evolved independently in these felid species.

Microglial analysis in whole‐mount retina of Alzheimer's disease 3xTg‐AD mouse model

AbstractPurposeAlzheimer’s Disease (AD) is a primary degenerative disease that is characterized by the accumulation of β‐amyloid and tau in central nervous system, even in visual brain areas and in the retina. This raises the possibility that tracking changes in the retina can be used to assess neurodegeneration in AD.MethodsWe analysed the retinas of 16‐month‐old 3xTg‐AD transgenic mice, which are used as an animal model of AD, and wild‐type (C57BL6/129S). Whole mount retinas were stained with antibodies against Iba‐1 and OX‐6. The morphology, number and soma size of microglia in the retina were analysed.ResultsRetinas from 3xTg‐AD transgenic mice showed signs of microglial activation relative to retinas from wild‐type animals: greater number of microglia, thickening of microglial soma, retraction of microglial processes and migration of microglia within and between layers. This activation was associated with alterations in the microglial plexus in the external plexiform layer and in the complex of internal layers. Retinas from 3xTg‐AD mice showed circular groups of microglia that reoriented and polarized their processes differently than in control retinas. Moreover, it was found a significant increase in both the number and cell body area of Iba‐1+ cells in 3xTg‐AD retinas in comparison with wild‐type retinas.ConclusionsRetinas from a mouse model of AD show signs of microglial activation. Reactive gliosis caused by AD‐associated protein aggregation may trigger morphological changes in the retinas of AD patients.AcknowledgmentsSanta Casa Mantero Belard Award 2015 (MB‐1049‐2015), Foundation for Science and Technology (PEst UID/NEU/04539/2013), COMPETE‐FEDER (POCI‐01‐0145‐FEDER‐007440), and Centro 2020 Regional Operational Programme (CENTRO‐01‐0145‐FEDER‐000008: BrainHealth 2020).

Overlapping morphological and functional properties between M4 and M5 intrinsically photosensitive retinal ganglion cells.

Multiple retinal ganglion cell (RGC) types in the mouse retina mediate pattern vision by responding to specific features of the visual scene. The M4 and M5 melanopsin-expressing, intrinsically photosensitive retinal ganglion cell (ipRGC) subtypes are two RGC types that are thought to play major roles in pattern vision. The M4 ipRGCs overlap in population with ON-alpha RGCs, while M5 ipRGCs were recently reported to exhibit opponent responses to different wavelengths of light (color opponency). Despite their seemingly distinct roles in visual processing, previous reports have suggested that these two populations may exhibit overlap in their morphological and functional properties, which calls into question whether these are in fact distinct RGC types. Here, we show that M4 and M5 ipRGCs are distinct morphological classes of ipRGCs, but they cannot be exclusively differentiated based on color opponency and dendritic morphology as previously reported. Instead, we find that M4 and M5 ipRGCs can only be distinguished based on soma size and the number of dendritic branch points in combination with SMI-32 immunoreactivity. These results have important implications for clearly defining RGC types and their roles in visual behavior.

Inhibition of excessive kallikrein-8 improves neuroplasticity in Alzheimer's disease mouse model

We recently identified excessive cerebral kallikrein-8 (KLK8) mRNA and protein levels at incipient stages of Alzheimer's disease (AD) in AD patients and TgCRND8 mice. Additionally, we showed that antibody-mediated KLK8 inhibition exerts therapeutic effects on AD along with enhancing neuroplasticity, resulting in improved spatial memory in mice. Mounting evidence further substantiates an important role of the protease KLK8 in neuroplasticity. In the present study we sought to gain new mechanistic insights in the interplay between KLK8, neuroplasticity and tau phosphorylation in the context of AD. We here demonstrate that KLK8 inhibition increased the number of hippocampal Ki-67 and doublecortin positive, proliferative neuronal progenitor cells in transgenic mice, whereas the same action in wildtypes had no effect. In line with these results, KLK8 inhibition reduced the levels of its pro-proliferative interaction partners KLK6 and protease-activated receptor 2 only in wildtypes, while the levels of its proliferation-supporting substrate neuregulin-1 and the non-complexed form of its complexing-partner phosphatidylethanolamine binding protein 1 were enhanced in both genotypes. Concomitant incubation of beta-amyloid (Aβ)-producing primary neurons with KLK8 and its inhibitory antibody increased neurite complexity and soma size. KLK8 inhibition in SH-SY5Y cells or in primary neurons increased levels of the neuroplasticity-supporting KLK8 substrate ephrin receptor B2 (EPHB2) and total tau while decreasing the relative amount of phospho-tau in relation to total tau. KLK8 blockade further enhanced cell proliferation in SH-SY5Y cells. Additional co-incubation with an inhibitory anti-EPHB2 antibody decreased total tau levels and neurite complexity and increased the ratio of phospho-tau/total tau, underlining the key role of EPHB2 on this plastic change. In a reverse in vitro approach, KLK8 induction reduced EPHB2 and total tau and increased the ratio of phospho-tau/total tau, leading to impaired proliferation and neuronal differentiation. These results underline the therapeutic potential of KLK8 inhibition by counteracting plasticity deficits in AD-affected brain.

Behavioral recovery and spinal motoneuron remodeling after polyethylene glycol fusion repair of singly cut and ablated sciatic nerves.

Polyethylene glycol repair (PEG-fusion) of severed sciatic axons restores their axoplasmic and membrane continuity, prevents Wallerian degeneration, maintains muscle fiber innervation, and greatly improves recovery of voluntary behaviors. We examined alterations in spinal connectivity and motoneuron dendritic morphology as one potential mechanism for improved behavioral function after PEG-fusion. At 2–112 days after a single-cut or allograft PEG-fusion repair of transected or ablated sciatic nerves, the number, size, location, and morphology of motoneurons projecting to the tibialis anterior muscle were assessed by retrograde labeling. For both lesion types, labeled motoneurons were found in the appropriate original spinal segment, but also in inappropriate segments, indicating mis-pairings of proximal-distal segments of PEG-fused motor axons. Although the number and somal size of motoneurons was unaffected, dendritic distributions were altered, indicating that PEG-fusion preserves spinal motoneurons but reorganizes their connectivity. This spinal reorganization may contribute to the remarkable behavioral recovery seen after PEG-fusion repair.

Selective-cold output through a distinct subset of lamina I spinoparabrachial neurons.

Spinal projection neurons are a major pathway through which somatic stimuli are conveyed to the brain. However, the manner in which this information is coded is poorly understood. Here, we report the identification of a modality-selective spinoparabrachial (SPB) neuron subtype with unique properties. Specifically, we find that cold-selective SPB neurons are differentiated by selective afferent input, reduced sensitivity to substance P, distinct physiological properties, small soma size, and low basal drive. In addition, optogenetic experiments reveal that cold-selective SPB neurons do not receive input from Nos1 inhibitory interneurons and, compared with other SPB neurons, show significantly smaller inhibitory postsynaptic currents upon activation of Pdyn inhibitory interneurons. Together, these data suggest that cold output from the spinal cord to the parabrachial nucleus is mediated by a specific cell type with distinct properties.

Size-Dependent Vulnerability of Lumbar Motor Neuron Dendritic Degeneration in SOD1G93A Mice.

The motor neuron (MN) soma surface area is correlated with motor unit type. Larger MNs innervate fast fatigue-intermediate (FInt) or fast-fatiguable (FF) muscle fibers in type FInt and FF motor units, respectively. Smaller MNs innervate slow-twitch fatigue-resistant (S) or fast fatigue-resistant (FR) muscle fibers in type S and FR motor units, respectively. In amyotrophic lateral sclerosis (ALS), FInt and FF motor units are more vulnerable, with denervation and MN death occurring for these units before the more resilient S and FR units. Abnormal MN dendritic arbors have been observed in ALS in humans and rodent models. We used a Golgi-Cox impregnation protocol to examine soma size-dependent changes in the dendritic morphology of lumbar MNs in SOD1G93A mice, a model of ALS, at pre-symptomatic, onset and mid-disease stages. In wildtype control mice, the relationship between MN soma surface area and dendritic length or dendritic spine number was highly linear (i.e., increased MN soma size correlated with increased dendritic length and spines). By contrast, in SOD1G93A mice, this linear relationship was lost and dendritic length reduction and spine loss were observed in larger MNs, from pre-symptomatic stages onward. These changes correlated with the neuromotor symptoms of ALS in rodent models. At presymptomatic ages, changes were restricted to the larger MNs, likely to comprise vulnerable FInt and FF motor units. Our results suggest morphological changes of MN dendrites and dendritic spines are likely to contribute ALS pathogenesis, not compensate for it. Anat Rec, 303:1455-1471, 2020. © 2019 American Association for Anatomy.

Scaling of the AIS and Somatodendritic Compartments in α S RGCs.

The anatomical properties of the axon initial segment (AIS) are tailored in certain types of CNS neurons to help optimize different aspects of neuronal function. Here, we questioned whether the AISs of retinal ganglion cells (RGC) were similarly customized, and if so, whether they supported specific RGC functions. To explore this, we measured the AIS properties in alpha sustained RGCs (α S RGCs) of mouse; α S RGCs sizes vary systematically along the nasal temporal axis of the retina, making these cells an attractive population with which to study potential correlations between AIS properties and cell size. Measurements of AIS length as well as distance from the soma revealed that both were scaled to cell size, i.e., cells with large dendritic fields had long AISs that were relatively far from the soma. Within the AIS, the percentage of Nav1.6 voltage-gated sodium channels remained highly consistent, regardless of cell size or other AIS properties. Although ON RGCs were slightly larger than OFF cells at any given location of the retina, the level of scaling and relative distribution of voltage-gated sodium channels were highly similar. Computational modeling revealed that AIS scaling influenced spiking thresholds, spike rate as well as the kinetics of individual action potentials, Interestingly, the effect of individual features of the AIS varied for different neuronal functions, e.g., AIS length had a larger effect on the efficacy by which the AIS initiated spike triggered the somatic spike than it did on repetitive spiking. The polarity of the effect varied for different properties, i.e., increases to soma size increased spike threshold while increases to AIS length decreased threshold. Thus, variations in the relative level of scaling for individual components could fine tune threshold or other neuronal functions. Light responses were highly consistent across the full range of cell sizes suggesting that scaling may post-synaptically shape response stability, e.g., in addition to several well-known pre-synaptic contributors.

Role of Delayed Neuroglial Activation in Impaired Cerebral Blood Flow Restoration Following Comorbid Injury.

Besides other causes, ischemia and Alzheimer's disease pathology is also linked to decreased cerebral blood flow (CBF). There is little or no consensus about the role of neuroglial cells in maintaining CBF in various neuropathologies. This consensus becomes scarcer when it comes to clinical and experimental cases of comorbid Abeta-amyloid (Aβ) toxicity and ischemia. Here, a comorbid rat model of Aβ toxicity and endothelin-1 induced ischemia (ET1) not only demonstrated the appearance of axotomized phagocytosed pyknotic neurons (NeuN) immediately after the injury, but also showed a diversity of continuously changing neuroglia (MHC Class II/OX6, Iba1) and macrophage (Iba1/CD68) phenotypes with round, stout somas, and retracted processes. This is indicative of a response to a concomitant increase in large fluid-filled spaces due to the vascular leakage. Ironically 4 weeks after the injury despite a conclusive reduction in neurons, CBF restoration in ET1 rats was associated with a massive increase in neuroglial cell numbers, hypertrophy, ramification, and soma sizes bordering the continuously reducing lesion core and inflamed vasculature, possibly to shield their leaky phenotype. Astrocytes were also found to be releasing matrix metalloproteinase9 (MMP9), which stabilized matrix ligand β-dystroglycan (β-DG) in repaired or functional vessels. Changing neuroglia phenotypes, responses, motility, astrocytic recruitment of MMP9, and β-DG stabilization implies the role of communication between neuroglia and endothelium in recovering CBF, in the absence of neurons, in ET1 rats compared to Aβ+ET1 rats, which showed characteristics delayed neuroglial activation. Stimulation of timely neuroglial reactivity may serve as a viable strategy to compensate for the neuronal loss in restoring CBF in comorbid cases of ischemia and Aβ toxicity.

Modification of Peak Plasticity Induced by Brief Dark Exposure.

The capacity for neural plasticity in the mammalian central visual system adheres to a temporal profile in which plasticity peaks early in postnatal development and then declines to reach enduring negligible levels. Early studies to delineate the critical period in cats employed a fixed duration of monocular deprivation to measure the extent of ocular dominance changes induced at different ages. The largest deprivation effects were observed at about 4 weeks postnatal, with a steady decline in plasticity thereafter so that by about 16 weeks only small changes were measured. The capacity for plasticity is regulated by a changing landscape of molecules in the visual system across the lifespan. Studies in rodents and cats have demonstrated that the critical period can be altered by environmental or pharmacological manipulations that enhance plasticity at ages when it would normally be low. Immersion in complete darkness for long durations (dark rearing) has long been known to alter plasticity capacity by modifying plasticity-related molecules and slowing progress of the critical period. In this study, we investigated the possibility that brief darkness (dark exposure) imposed just prior to the critical period peak can enhance the level of plasticity beyond that observed naturally. We examined the level of plasticity by measuring two sensitive markers of monocular deprivation, namely, soma size of neurons and neurofilament labeling within the dorsal lateral geniculate nucleus. Significantly larger modification of soma size, but not neurofilament labeling, was observed at the critical period peak when dark exposure preceded monocular deprivation. This indicated that the natural plasticity ceiling is modifiable and also that brief darkness does not simply slow progress of the critical period. As an antecedent to traditional amblyopia treatment, darkness may increase treatment efficacy even at ages when plasticity is at its highest.

TRPM8-mediated cutaneous stimulation modulates motor neuron activity during treadmill stepping in mice

Motor units are generally recruited from the smallest to the largest following the size principle, while cutaneous stimulation has the potential to affect spinal motor control. We aimed to examine the effects of stimulating transient receptor potential channel sub-family M8 (TRPM8) combined with exercise on the modulation of spinal motor neuron (MN) excitability. Mice were topically administrated 1.5% icilin on the hindlimbs, followed by treadmill stepping. Spinal cord sections were immunostained with antibodies against c-fos and choline acetyltransferase. Icilin stimulation did not change the number of c-fos+ MNs, but increased the average soma size of the c-fos+ MNs during low-speed treadmill stepping. Furthermore, icilin stimulation combined with stepping increased c-fos+ cholinergic interneurons near the central canal, which are thought to modulate MN excitability. These findings suggest that TRPM8-mediated cutaneous stimulation with low-load exercise promotes preferential recruitment of large MNs and is potentially useful as a new training method for rehabilitation.

Brain microglial activation in chronic pain-associated affective disorder

A growing body of evidence from both clinical and animal studies indicates that chronic neuropathic pain is associated with comorbid affective disorders. Spinal cord microglial activation is involved in nerve injury-induced pain hypersensitivity characterizing neuropathic pain. However, there is a lack of thorough assessments of microglial activation in the brain after nerve injury. In the present study, we characterized microglial activation in brain sub-regions of CX3CR1GFP/+ mice after chronic constriction injury (CCI) of the sciatic nerve, including observations at delayed time points when affective brain dysfunctions such as depressive-like behaviors typically develop. Mice manifested chronic mechanical hypersensitivity immediately after CCI and developed depressive-like behaviors 8 weeks post-injury. Concurrently, significant increases of soma size and microglial cell number were observed in the medial prefrontal cortex (mPFC), hippocampus, and amygdala 8 weeks post-injury. Transcripts of CD11b, and TNF-α, genes associated with microglial activation or depressive-like behaviors, are correspondingly upregulated in these brain areas. Our results demonstrate that microglia are activated in specific brain sub-regions after CCI at delayed time points and imply that brain microglial activation plays a role in chronic pain-associated affective disorders.

Neuroprotective and Anti-Inflammatory Effects of a Hydrophilic Saffron Extract in a Model of Glaucoma.

Glaucoma is a neurodegenerative disease characterized by the loss of retinal ganglion cells (RGCs). An increase in the intraocular pressure is the principal risk factor for such loss, but controlling this pressure does not always prevent glaucomatous damage. Activation of immune cells resident in the retina (microglia) may contribute to RGC death. Thus, a substance with anti-inflammatory activity may protect against RGC degeneration. This study investigated the neuroprotective and anti-inflammatory effects of a hydrophilic saffron extract standardized to 3% crocin content in a mouse model of unilateral, laser-induced ocular hypertension (OHT). Treatment with saffron extract decreased microglion numbers and morphological signs of their activation, including soma size and process retraction, both in OHT and in contralateral eyes. Saffron extract treatment also partially reversed OHT-induced down-regulation of P2RY12. In addition, the extract prevented retinal ganglion cell death in OHT eyes. Oral administration of saffron extract was able to decrease the neuroinflammation associated with increased intraocular pressure, preventing retinal ganglion cell death. Our findings indicate that saffron extract may exert a protective effect in glaucomatous pathology.

Activation of epiplexus macrophages in hydrocephalus caused by subarachnoid hemorrhage and thrombin.

AimsWe have found that hydrocephalus development in spontaneously hypertensive rats was associated with activation of epiplexus cells. The current study examined whether epiplexus cell activation occurs in a rat subarachnoid hemorrhage (SAH), whether activation would be greater in a subset of rats that developed hydrocephalus and the potential role of thrombin in epiplexus cell activation.MethodsThere were two parts in this study. First, an endovascular perforation was performed in rats to induce SAH. Second, rats received an intraventricular infusion of either thrombin or saline. Magnetic resonance imaging was used to measure the ventricular volumes. Immunofluorescence and immunohistochemistry were used to study epiplexus cell activation.ResultsIba‐1, OX‐6, and CD68 were expressed in the epiplexus cells of the choroid plexus in sham‐operated rats. SAH increased Iba‐1 and CD68 immunoreactivity in epiplexus cells in addition to an increase in Iba‐1‐positive cell soma size. Those effects were greater in rats that developed hydrocephalus. Intraventricular thrombin mimicked the effects of SAH on epiplexus cell activation and hydrocephalus.ConclusionThis study supports the concept that epiplexus cell activation is associated with hydrocephalus development. Epiplexus cell activation may be in response to thrombin production after hemorrhage, and it may be a therapeutic target.

Lysophosphatidic acid receptor 1 (LPA1) plays critical roles in microglial activation and brain damage after transient focal cerebral ischemia

BackgroundLysophosphatidic acid receptor 1 (LPA1) is in the spotlight because its synthetic antagonist has been under clinical trials for lung fibrosis and psoriasis. Targeting LPA1 might also be a therapeutic strategy for cerebral ischemia because LPA1 triggers microglial activation, a core pathogenesis in cerebral ischemia. Here, we addressed this possibility using a mouse model of transient middle cerebral artery occlusion (tMCAO).MethodsTo address the role of LPA1 in the ischemic brain damage, we used AM095, a selective LPA1 antagonist, as a pharmacological tool and lentivirus bearing a specific LPA1 shRNA as a genetic tool. Brain injury after tMCAO challenge was accessed by determining brain infarction and neurological deficit score. Role of LPA1 in tMCAO-induced microglial activation was ascertained by immunohistochemical analysis. Proinflammatory responses in the ischemic brain were determined by qRT-PCR and immunohistochemical analyses, which were validated in vitro using mouse primary microglia. Activation of MAPKs and PI3K/Akt was determined by Western blot analysis.ResultsAM095 administration immediately after reperfusion attenuated brain damage such as brain infarction and neurological deficit at 1 day after tMCAO, which was reaffirmed by LPA1 shRNA lentivirus. AM095 administration also attenuated brain infarction and neurological deficit at 3 days after tMCAO. LPA1 antagonism attenuated microglial activation; it reduced numbers and soma size of activated microglia, reversed their morphology into less toxic one, and reduced microglial proliferation. Additionally, LPA1 antagonism reduced mRNA expression levels of proinflammatory cytokines and suppressed NF-κB activation, demonstrating its regulatory role of proinflammatory responses in the ischemic brain. Particularly, these LPA1-driven proinflammatory responses appeared to occur in activated microglia because NF-κB activation occurred mainly in activated microglia in the ischemic brain. Regulatory role of LPA1 in proinflammatory responses of microglia was further supported by in vitro findings using lipopolysaccharide-stimulated cultured microglia, showing that suppressing LPA1 activity reduced mRNA expression levels of proinflammatory cytokines. In the ischemic brain, LPA1 influenced PI3K/Akt and MAPKs; suppressing LPA1 activity decreased MAPK activation and increased Akt phosphorylation.ConclusionThis study demonstrates that LPA1 is a new etiological factor for cerebral ischemia, strongly indicating that its modulation can be a potential strategy to reduce ischemic brain damage.

Discriminative Stimulus and Low Abuse Liability Effects of Novel Endomorphin Analogs Suggest a Potential Treatment Indication for Opioid Use Disorder.

Opioid dependence can be difficult to manage using existing pharmacotherapies. A long-acting opioid with low abuse liability that substitutes for a shorter-acting opioid may improve treatment of opioid use disorders (OUDs). We recently characterized an endomorphin (EM) analog (ZH853) that produced a longer duration of antinociception compared with morphine, but did not produce self-administration or several other adverse effects preclinically. Here, we further characterized ZH853 in tests of antinociception, abuse liability, and drug discrimination. A conditioned place preference (CPP) procedure, that included a locomotor activity assessment, was used to test abuse liability in rats. Subsequently, dopamine (DA) cell-somas located in the ventral tegmental area (VTA) from these rats were assessed by size using immunohistochemistry and Stereo Investigator software. A hot-plate antinociception test in male and female mice confirmed central penetration. Morphine-substitution effects of several EM analogs (ZH850, ZH831, and ZH853) were tested in a drug discrimination (DD) procedure in rats. Morphine produced dose-dependent CPP and locomotor sensitization and reduced the size of DA cell somas in VTA, whereas ZH853 did not produce any of these effects relative to control. The antinociceptive effects of ZH853 were μ-receptor selective since β-funaltrexamine antagonized these effects. Rats responded on a morphine-trained lever when injected with ZH831 and ZH853 during DD experiments. The favorable morphine-substitution effects of these EM analogs relative to their low abuse liability indicate promising novel compounds that may improve treatment of OUD. SIGNIFICANCE STATEMENT: In this experiment, we investigated the preclinical effects of novel endomorphin analogs for use as substitution therapies for opioid use disorder, a problem that has contributed to an opioid overdose epidemic. Several endomorphin analogs substituted for morphine without producing adverse effects, including reward behaviors associated with abuse liability. These compounds have the potential to become important additional tools to treat opioid use disorders.

Segmentation, Tracing, and Quantification of Microglial Cells from 3D Image Stacks

Microglia play a central role in modulating synaptic structure and physiology, learning and memory processes. They exhibit morphological changes to perform these roles, therefore the morphological study of microglia can help to understand their functionality. Many promising methods are proposed to automatically segment the blood vessels or reconstruct the neuronal morphology. However, they often fail to accurately capture microglia organizations due to the striking structural differences. This requires a more sophisticated approach of reconstruction taking into account the varying nature of branch structures and soma sizes. To this end, we propose an automated method to reconstruct microglia, and quantify their features from 2D/3D image datasets. We first employ multilevel thresholding to segment soma volumes(3D)/areas(2D) and recognize foreground voxels/pixels. Seed points sampled from the foreground, are connected to form the skeleton of the branches via the tracing process. The reconstructed data is quantified and written in SWC standard file format. We have applied our method to 3D image datasets of microglia, then evaluated the results using ground truth data, and compared them to those achieved via the state-of-the-art methods. Our method outperforms the others both in accuracy and computational time.

Relationship among ETV1 genetic polymorphisms, PFS, and microRNA in gastrointestinal stromal tumors.

e22513 Background: In order to evaluate the pharmacokinetic and pharmacogenomic determinants for prognosis of gastrointestinal stromal tumor (GIST). It is necessary to explore a biological predictors to predict and optimal therapeutic strategy. But there were few studied conducted to explore the germline mutation and its mechanisms. Methods: A total of 75 GIST patients treated with Imatinib were enrolled. 35 SNPs (single nucleotide polymorphisms) in KIT/ PDGFRA/ PDGFRB/ ETV1/ FLT1/ MAPK1 et. al were detected using Agena Massarray matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) platform. COX regression analyses were performed to evaluate the key factors of PFS. The luciferase reporter system of rs3735343 wild type and mutation were established. The GIST-T1 cell line was used to evaluate the biology effect of rs3735343. This study was approved by the ethical committee of Sun Yat-Sen University Cancer Center. Results: Several factors influence the PFS, including KIT somatic mutation, tumor size and germline mutation ABCC4 rs4148551, ETV1 rs3735343, FLT1 rs3751397, KIT rs3822214. And COX regression showed that rs3735343 and tumor size are associated with PFS (P = 0.009 and 0.032, Risk Ratio = 8.995 and 4.173). And we found that rs3735343 mutation type can regulate ETV1 3’UTR and protein expression level through miR-4311 in vitro. Conclusions: The primary determinants of PFS in this somatic and germline mutation model suggested new biomarkers and different mechanisms involved in prognosis. This is the first report showed that ETV1 genetic polymorphisms may influence the prognosis through miRNA-4311. Targeting ETV1, miRNA-4311 or their relative pathway might be a new therapy strategy.

Chronic mTORC1 inhibition rescues behavioral and biochemical deficits resulting from neuronal Depdc5 loss in mice.

DEPDC5 is now recognized as one of the genes most often implicated in familial/inherited focal epilepsy and brain malformations. Individuals with pathogenic variants in DEPDC5 are at risk for epilepsy, associated neuropsychiatric comorbidities and sudden unexplained death in epilepsy. Depdc5flox/flox-Syn1Cre (Depdc5cc+) neuronal-specific Depdc5 knockout mice exhibit seizures and neuronal mTORC1 hyperactivation. It is not known if Depdc5cc+ mice have a hyperactivity/anxiety phenotype, die early from terminal seizures or whether mTOR inhibitors rescue DEPDC5-related seizures and associated comorbidities. Herein, we report that Depdc5cc+ mice were hyperactive in open-field testing but did not display anxiety-like behaviors on the elevated-plus maze. Unlike many other mTOR-related models, Depdc5cc+ mice had minimal epileptiform activity and rare seizures prior to seizure-induced death, as confirmed by video-EEG monitoring. Treatment with the mTORC1 inhibitor rapamycin starting after 3weeks of age significantly prolonged the survival of Depdc5cc+ mice and partially rescued the behavioral hyperactivity. Rapamycin decreased the enlarged brain size of Depdc5cc+ mice with corresponding decrease in neuronal soma size. Loss of Depdc5 led to a decrease in the other GATOR1 protein levels (NPRL2 and NPRL3). Rapamycin failed to rescue GATOR1 protein levels but rather rescued downstream mTORC1 hyperactivity as measured by phosphorylation of S6. Collectively, our data provide the first evidence of behavioral alterations in mice with Depdc5 loss and support mTOR inhibition as a rational therapeutic strategy for DEPDC5-related epilepsy in humans.