Unraveling the transcriptional features and gene expression networks of pathogenic and saprotrophic Ophiostoma species during the infection of Ulmus americana.

American elm (Ulmus americana), highly prized for its ornamental value, has suffered two successive outbreaks of Dutch elm disease (DED) caused by ascomycete fungi belonging to the genus Ophiostoma. To identify the genes linked to the pathogenicity of different species and lineages of Ophiostoma, we inoculated 2-year-old U. americana saplings with six strains representing three species of DED fungi, and one strain of the saprotroph Ophiostoma quercus. Differential expression analyses were performed following RNA sequencing of fungal transcripts recovered at 3- and 10-days post-infection. Based on a total of 8,640 Ophiostoma genes, we observed a difference in fungal gene expression depending on the strain inoculated and the time of incubation in host tissue. Some genes overexpressed in the more virulent strains of Ophiostoma encode hydrolases that possibly act synergistically. A mutant of Ophiostoma novo-ulmi in which the gene encoding the ogf1 transcription factor had been deleted did not produce transcripts for the gene encoding the hydrophobin cerato-ulmin and was less virulent. Weighted gene correlation network analyses identified several candidate pathogenicity genes distributed among 13 modules of interconnected genes.IMPORTANCEOphiostoma is a genus of cosmopolitan fungi that belongs to the family Ophiostomataceae and includes the pathogens responsible for two devastating pandemics of Dutch elm disease (DED). As the mechanisms of action of DED agents remain unclear, we carried out the first comparative transcriptomic study including representative strains of the three Ophiostoma species causing DED, along with the phylogenetically close saprotrophic species Ophiostoma quercus. Statistical analyses of the fungal transcriptomes recovered at 3 and 10 days following infection of Ulmus americana saplings highlighted several candidate genes associated with virulence and host-pathogen interactions wherein each strain showed a distinct transcriptome. The results of this research underscore the importance of investigating the transcriptional behavior of different fungal taxa to understand their pathogenicity and virulence in relation to the timeline of infection.

Chronic pregabalin treatment protects against spreading depolarization and alters hippocampal synaptic characteristics in a model of familial hemiplegic migraine-type 1.

Familial hemiplegic migraine type-1 (FHM-1) is a form of migraine with aura caused by mutations in the P/Q-type (Cav2.1) voltage-gated calcium channel. Pregabalin, used clinically in the treatment of chronic pain and epilepsy, inhibits P/Q-type calcium channel activity and recent studies suggest that it may have potential for the treatment of migraine. Spreading Depolarization (SD) is a neurophysiological phenomenon that can occur during migraine with aura by propagating a wave of silenced neuronal function through cortex and sometimes subcortical brain structures. Here, utilizing an optogenetic stimulation technique optimized to allow for non-invasive initiation of cortical SD, we demonstrate that chronic pregabalin administration [12 mg/kg/day (s.c.)] in vivo increased the threshold for cortical spreading depolarization in transgenic mice harboring the clinically-relevant Cav2.1S218L mutation (S218L). In addition, chronic pregabalin treatment limited subcortical propagation of recurrent spreading depolarization events to the striatum and hippocampus in both wild-type and S218L mice. To examine contributing underlying mechanisms of action of chronic pregabalin, we performed whole-cell patch-clamp electrophysiology in CA1 neurons in ex vivo brain slices from mice treated with chronic pregabalin vs vehicle. In WT mice, chronic pregabalin produced a decrease in spontaneous excitatory postsynaptic current (sEPSC) amplitude with no effect on frequency. In contrast, in S218L mice chronic pregabalin produced an increase in sEPSC amplitude and decreased frequency. These electrophysiological findings suggest that in FHM-1 mice chronic pregabalin acts through both pre- and post-synaptic mechanisms in CA1 hippocampal neurons to elicit FHM-1 genotype-specific inhibitory action. The results highlight the potential of chronic pregabalin to limit recurrent SD to subcortical brain structures during pathophysiological events in both the genetically-normal and FHM-1 brain. The work further provides insights into FHM-1 pathophysiology and the potential for chronic pregabalin treatment to prevent SD in migraineurs.

Pannexin-1 opening in neuronal edema causes cell death but also leads to protection via increased microglia contacts.

Neuronal swelling during cytotoxic edema is triggered by Na+ and Cl- entry and is Ca2+ independent. However, the causes of neuronal death during swelling are unknown. Here, we investigate the role of large-conductance Pannexin-1 (Panx1) channels in neuronal death during cytotoxic edema. Panx1 channel inhibitors reduce and delay neuronal death in swelling triggered by voltage-gated Na+ entry with veratridine. Neuronal swelling causes downstream production of reactive oxygen species (ROS) that opens Panx1 channels. We confirm that ROS activates Panx1 currents with whole-cell electrophysiology and find scavenging ROS is neuroprotective. Panx1 opening and subsequent ATP release attract microglial processes to contact swelling neurons. Depleting microglia using the CSF1 receptor antagonist PLX3397 or blocking P2Y12 receptors exacerbates neuronal death, suggesting that the Panx1-ATP-dependent microglia contacts are neuroprotective. We conclude that cytotoxic edema triggers oxidative stress in neurons that opens Panx1 to trigger death but also initiates neuroprotective feedback mediated by microglia contacts.

Complexities underlying the breeding and deployment of Dutch elm disease resistant elms.

Dutch elm disease (DED) is a vascular wilt disease caused by the pathogens Ophiostoma ulmi and Ophiostoma novo-ulmi with multiple ecological phases including pathogenic (xylem), saprotrophic (bark) and vector (beetle flight and beetle feeding wound) phases. Due to the two DED pandemics during the twentieth century the use of elms in landscape and forest restoration has declined significantly. However new initiatives for elm breeding and restoration are now underway in Europe and North America. Here we discuss complexities in the DED 'system' that can lead to unintended consequences during elm breeding and some of the wider options for obtaining durability or 'field resistance' in released material, including (1) the phenotypic plasticity of disease levels in resistant cultivars infected by O. novo-ulmi; (2) shortcomings in test methods when selecting for resistance; (3) the implications of rapid evolutionary changes in current O. novo-ulmi populations for the choice of pathogen inoculum when screening; (4) the possibility of using active resistance to the pathogen in the beetle feeding wound, and low attractiveness of elm cultivars to feeding beetles, in addition to resistance in the xylem; (5) the risk that genes from susceptible and exotic elms be introgressed into resistant cultivars; (6) risks posed by unintentional changes in the host microbiome; and (7) the biosecurity risks posed by resistant elm deployment. In addition, attention needs to be paid to the disease pressures within which resistant elms will be released. In the future, biotechnology may further enhance our understanding of the various resistance processes in elms and our potential to deploy trees with highly durable resistance in elm restoration. Hopefully the different elm resistance processes will prove to be largely under durable, additive, multigenic control. Elm breeding programmes cannot afford to get into the host-pathogen arms races that characterise some agricultural host-pathogen systems.

Comparative Analysis of Transcriptomes of Ophiostoma novo-ulmi ssp. americana Colonizing Resistant or Sensitive Genotypes of American Elm.

The Ascomycete Ophiostoma novo-ulmi threatens elm populations worldwide. The molecular mechanisms underlying its pathogenicity and virulence are still largely uncharacterized. As part of a collaborative study of the O. novo-ulmi-elm interactome, we analyzed the O. novo-ulmi ssp. americana transcriptomes obtained by deep sequencing of messenger RNAs recovered from Ulmus americana saplings from one resistant (Valley Forge, VF) and one susceptible (S) elm genotypes at 0 and 96 h post-inoculation (hpi). Transcripts were identified for 6424 of the 8640 protein-coding genes annotated in the O. novo-ulmi nuclear genome. A total of 1439 genes expressed in planta had orthologs in the PHI-base curated database of genes involved in host-pathogen interactions, whereas 472 genes were considered differentially expressed (DEG) in S elms (370 genes) and VF elms (102 genes) at 96 hpi. Gene ontology (GO) terms for processes and activities associated with transport and transmembrane transport accounted for half (27/55) of GO terms that were significantly enriched in fungal genes upregulated in S elms, whereas the 22 GO terms enriched in genes overexpressed in VF elms included nine GO terms associated with metabolism, catabolism and transport of carbohydrates. Weighted gene co-expression network analysis identified three modules that were significantly associated with higher gene expression in S elms. The three modules accounted for 727 genes expressed in planta and included 103 DEGs upregulated in S elms. Knockdown- and knockout mutants were obtained for eight O. novo-ulmi genes. Although mutants remained virulent towards U. americana saplings, we identified a large repertoire of additional candidate O. novo-ulmi pathogenicity genes for functional validation by loss-of-function approaches.

Age-dependent gray matter demyelination is associated with leptomeningeal neutrophil accumulation.

People living with multiple sclerosis (MS) experience episodic CNS white matter lesions instigated by autoreactive T cells. With age, patients with MS show evidence of gray matter demyelination and experience devastating nonremitting symptomology. What drives progression is unclear and studying this has been hampered by the lack of suitable animal models. Here, we show that passive experimental autoimmune encephalomyelitis (EAE) induced by an adoptive transfer of young Th17 cells induced a nonremitting clinical phenotype that was associated with persistent leptomeningeal inflammation and cortical pathology in old, but not young, SJL/J mice. Although the quantity and quality of T cells did not differ in the brains of old versus young EAE mice, an increase in neutrophils and a decrease in B cells were observed in the brains of old mice. Neutrophils were also found in the leptomeninges of a subset of progressive MS patient brains that showed evidence of leptomeningeal inflammation and subpial cortical demyelination. Taken together, our data show that while Th17 cells initiate CNS inflammation, subsequent clinical symptoms and gray matter pathology are dictated by age and associated with other immune cells, such as neutrophils.

Hyperexcitable superior colliculus and fatal brainstem spreading depolarization in a model of Sudden Unexpected Death in Epilepsy.

Cardiorespiratory arrest and death in mouse models of sudden unexpected death in epilepsy occur when spreading depolarization is triggered by cortical seizures and then propagates to the brainstem. However, the critical brain regions and the specific changes required to allow spreading depolarization to propagate to the brainstem under the relatively rare circumstances leading to a fatal seizure are unknown. We previously found that following cortical seizure-inducing electrical stimulation, spreading depolarization could occur in both the superior and inferior colliculi in Cacna1aS218L mice, but was never observed in wild-type animals or following non-seizure-inducing stimuli in Cacna1aS218L mice. Here, we show that optogenetic stimulation of the superior/inferior colliculi in Cacna1aS218L mice induces severe seizures, and resulting spreading depolarization in the superior/inferior colliculi that propagates to the brainstem and correlates with the respiratory arrest followed by cardiac arrest. Further, we show that neurons of the superior colliculus in Cacna1aS218L mice exhibit hyperexcitable properties that we propose underlie a distinct susceptibility to spreading depolarization. Our data suggest that the susceptibility of the superior colliculus to elicit fatal spreading depolarization is a result of either genetic or seizure-related alterations within the superior colliculus that may involve changes to structure, connectivity and/or excitability.

Deciphering the Genome-Wide Transcriptomic Changes during Interactions of Resistant and Susceptible Genotypes of American Elm with Ophiostoma novo-ulmi.

Dutch elm disease (DED), caused by Ophiostoma novo-ulmi (Onu), is a destructive disease of American elm (Ulmus americana L.). The molecular mechanisms of resistance and susceptibility against DED in American elm are still largely uncharacterized. In the present study, we performed a de novo transcriptome (RNA-sequencing; RNA-Seq) assembly of U. americana and compared the gene expression in a resistant genotype, 'Valley Forge', and a susceptible (S) elm genotype at 0 and 96 h post-inoculation of Onu. A total of 85,863 non-redundant unigenes were identified. Compared to the previously characterized U. minor transcriptome, U. americana has 35,290 similar and 55,499 unique genes. The transcriptomic variations between 'Valley Forge' and 'S' were found primarily in the photosynthesis and primary metabolism, which were highly upregulated in the susceptible genotype irrespective of the Onu inoculation. The resistance to DED was associated with the activation of RPM1-mediated effector-triggered immunity that was demonstrated by the upregulation of genes involved in the phenylpropanoids biosynthesis and PR genes. The most significantly enriched gene ontology (GO) terms in response to Onu were response to stimulus (GO:0006950), response to stress (GO:0050896), and secondary metabolic process (GO:0008152) in both genotypes. However, only in the resistant genotype, the defense response (GO:0006952) was among the topmost significantly enriched GO terms. Our findings revealed the molecular regulations of DED resistance and susceptibility and provide a platform for marker-assisted breeding of resistant American elm genotypes.

Gut microbes shape microglia and cognitive function during malnutrition.

Fecal-oral contamination promotes malnutrition pathology. Lasting consequences of early life malnutrition include cognitive impairment, but the underlying pathology and influence of gut microbes remain largely unknown. Here, we utilize an established murine model combining malnutrition and iterative exposure to fecal commensals (MAL-BG). The MAL-BG model was analyzed in comparison to malnourished (MAL mice) and healthy (CON mice) controls. Malnourished mice display poor spatial memory and learning plasticity, as well as altered microglia, non-neuronal CNS cells that regulate neuroimmune responses and brain plasticity. Chronic fecal-oral exposures shaped microglial morphology and transcriptional profile, promoting phagocytic features in MAL-BG mice. Unexpectedly, these changes occurred independently from significant cytokine-induced inflammation or blood-brain barrier (BBB) disruption, key gut-brain pathways. Metabolomic profiling of the MAL-BG cortex revealed altered polyunsaturated fatty acid (PUFA) profiles and systemic lipoxidative stress. In contrast, supplementation with an ω3 PUFA/antioxidant-associated diet (PAO) mitigated cognitive deficits within the MAL-BG model. These findings provide valued insight into the malnourished gut microbiota-brain axis, highlighting PUFA metabolism as a potential therapeutic target.

Independent Evolution Has Led to Distinct Genomic Signatures in Dutch Elm Disease-Causing Fungi and Other Vascular Wilts-Causing Fungal Pathogens.

Vascular wilts are important diseases caused by plant pathogenic fungi that result in the rapid death of their plant hosts. This is due to a systemic defense mechanism whereby the plant induces the compartmentalization of the infected vascular system in order to reduce the propagation of the fungus. The ascomycete class Sordariomycetes contains several species that cause vascular wilts in diverse plant hosts, and they can be classified into four taxonomic orders. The genetic mechanisms of pathogenesis have already been investigated in Fusarium and Verticillium species, but they have not yet been compared with other well-known wilt-causing species, especially fungi causing oak wilt or Dutch elm disease (DED). Here we analyzed 20 whole genome assemblies of wilt-causing fungi together with 56 other species using phylogenetic approaches to trace expansions and contractions of orthologous gene families and gene classes related to pathogenicity. We found that the wilt-causing pathogens evolved seven times, experiencing the largest fold changes in different classes of genes almost every time. However, some similarities exist across groups of wilt pathogens, particularly in Microascales and Ophiostomatales, and these include the common gains and losses of genes that make up secondary metabolite clusters (SMC). DED pathogens do not experience large-scale gene expansions, with most of the gene classes, except for some SMC families, reducing in number. We also found that gene family expansions in the most recent common ancestors of wilt pathogen groups are enriched for carbohydrate metabolic processes. Our study shows that wilt-causing species evolve primarily through distinct changes in their repertoires of pathogenicity-related genes and that there is the potential importance of carbohydrate metabolism genes for regulating osmosis in those pathogens that penetrate the plant vascular system.

An Efficient Strategy for Obtaining Mutants by Targeted Gene Deletion in Ophiostoma novo-ulmi.

The dimorphic fungus Ophiostoma novo-ulmi is the highly aggressive pathogen responsible for the current, highly destructive, pandemic of Dutch elm disease (DED). Genome and transcriptome analyses of this pathogen previously revealed that a large set of genes expressed during dimorphic transition were also potentially related to plant infection processes, which seem to be regulated by molecular mechanisms different from those described in other dimorphic pathogens. Then, O. novo-ulmi can be used as a representative species to study the lifestyle of dimorphic pathogenic fungi that are not shared by the "model species" Candida albicans and Ustilago maydis. In order to gain better knowledge of molecular aspects underlying infection process and symptom induction by dimorphic fungi that cause vascular wilt disease, we developed a high-throughput gene deletion protocol for O. novo-ulmi. The protocol is based on transforming a Δmus52 O. novo-ulmi mutant impaired for non-homologous end joining (NHEJ) as the recipient strain, and transforming this strain with the latest version of OSCAR plasmids. The latter are used for generating deletion constructs containing the toxin-coding Herpes simplex virus thymidine kinase (HSVtk) gene which prevents ectopic integration of the T-DNA in Ophiostoma DNA. The frequency of gene deletion by homologous recombination (HR) at the ade1 locus associated with purine nucleotide biosynthesis was up to 77.8% in the Δmus52 mutant compared to 2% in the wild-type (WT). To validate the high efficiency of our deletion gene methodology we deleted ade7, which also belongs to the purine nucleotide pathway, as well as bct2, ogf1, and opf2 which encode fungal binuclear transcription factors (TFs). The frequency of gene replacement by HR for these genes reached up to 94%. We expect that our methodology combining the use of NHEJ deficient strains and OSCAR plasmids will function with similar high efficiencies for other O. novo-ulmi genes and other filamentous fungi.

Location Matters: Navigating Regional Heterogeneity of the Neurovascular Unit.

The neurovascular unit (NVU) of the brain is composed of multiple cell types that act synergistically to modify blood flow to locally match the energy demand of neural activity, as well as to maintain the integrity of the blood-brain barrier (BBB). It is becoming increasingly recognized that the functional specialization, as well as the cellular composition of the NVU varies spatially. This heterogeneity is encountered as variations in vascular and perivascular cells along the arteriole-capillary-venule axis, as well as through differences in NVU composition throughout anatomical regions of the brain. Given the wide variations in metabolic demands between brain regions, especially those of gray vs. white matter, the spatial heterogeneity of the NVU is critical to brain function. Here we review recent evidence demonstrating regional specialization of the NVU between brain regions, by focusing on the heterogeneity of its individual cellular components and briefly discussing novel approaches to investigate NVU diversity.

Gamma frequency activation of inhibitory neurons in the acute phase after stroke attenuates vascular and behavioral dysfunction.

Alterations in gamma oscillations occur in several neurological disorders, and the entrainment of gamma oscillations has been recently proposed as a treatment for neurodegenerative disease. Optogenetic stimulation enhances recovery in models of stroke when applied weeks after injury; however, the benefits of acute brain stimulation have not been investigated. Here, we report beneficial effects of gamma-frequency modulation in the acute phase, within 1 h, after stroke. Transgenic VGAT-ChR2 mice are subject to awake photothrombotic stroke in an area encompassing the forelimb sensory and motor cortex. Optogenetic stimulation at 40 Hz in the peri-infarct zone recovers neuronal activity 24 h after stroke in motor and parietal association areas, as well as blood flow over the first week after stroke. Stimulation significantly reduces lesion volume and improves motor function. Our results suggest that acute-phase modulation of cortical oscillatory dynamics may serve as a target for neuroprotection against stroke.

Immunometabolism in the Brain: How Metabolism Shapes Microglial Function.

Immune cells react to their environment by flexibly reprogramming intracellular metabolic pathways that subsequently alter immune function, in a process called immunometabolism. However, in the CNS, the impact of metabolic reprogramming on microglia, neuroinflammation, and subsequently on brain function is poorly understood. As brain-resident macrophages, microglia are the CNS immune effectors and share similarities with peripheral immune cells. New tools for studying immunometabolism now allow the analysis of bioenergetic regulation with cellular resolution and, as a result, have uncovered previously unappreciated roles for microglial immunometabolism in shaping neuroinflammation. This review highlights evidence that microglia metabolism adapts to changes in brain energy homeostasis and that metabolic reprogramming regulates microglial polarization, thereby impacting pathological inflammatory responses in the brain.

Microglial metabolic flexibility supports immune surveillance of the brain parenchyma.

Microglia are highly motile cells that continuously monitor the brain environment and respond to damage-associated cues. While glucose is the main energy substrate used by neurons in the brain, the nutrients metabolized by microglia to support surveillance of the parenchyma remain unexplored. Here, we use fluorescence lifetime imaging of intracellular NAD(P)H and time-lapse two-photon imaging of microglial dynamics in vivo and in situ, to show unique aspects of the microglial metabolic signature in the brain. Microglia are metabolically flexible and can rapidly adapt to consume glutamine as an alternative metabolic fuel in the absence of glucose. During insulin-induced hypoglycemia in vivo or in aglycemia in acute brain slices, glutaminolysis supports the maintenance of microglial process motility and damage-sensing functions. This metabolic shift sustains mitochondrial metabolism and requires mTOR-dependent signaling. This remarkable plasticity allows microglia to maintain their critical surveillance and phagocytic roles, even after brain neuroenergetic homeostasis is compromised.

Hybridization and introgression drive genome evolution of Dutch elm disease pathogens.

Hybridization and the resulting introgression can drive the success of invasive species via the rapid acquisition of adaptive traits. The Dutch elm disease pandemics in the past 100 years were caused by three fungal lineages with permeable reproductive barriers: Ophiostoma ulmi, Ophiostoma novo-ulmi subspecies novo-ulmi and Ophiostoma novo-ulmi subspecies americana. Using whole-genome sequences and growth phenotyping of a worldwide collection of isolates, we show that introgression has been the main driver of genomic diversity and that it impacted fitness-related traits. Introgressions contain genes involved in host-pathogen interactions and reproduction. Introgressed isolates have enhanced growth rate at high temperature and produce different necrosis sizes on an in vivo model for pathogenicity. In addition, lineages diverge in many pathogenicity-associated genes and exhibit differential mycelial growth in the presence of a proxy of a host defence compound, implying an important role of host trees in the molecular and functional differentiation of these pathogens.

Nanoscale Surveillance of the Brain by Microglia via cAMP-Regulated Filopodia.

Microglia, the brain's immune cells, maintain homeostasis and sense pathological changes by continuously surveying the parenchyma with highly motile large processes. Here, we demonstrate that microglia also use thin actin-dependent filopodia that allow fast nanoscale sensing within discrete regions. Filopodia are distinct from large processes by their size, speed, and regulation mechanism. Increasing cyclic AMP (cAMP) by activating norepinephrine Gs-coupled receptors, applying nitric oxide, or inhibiting phosphodiesterases rapidly increases filopodia but collapses large processes. Alternatively, Gi-coupled P2Y12 receptor activation collapses filopodia but triggers large processes extension with bulbous tips. Similar control of cytoskeletal dynamics and microglial morphology by cAMP is observed in ramified primary microglia, suggesting that filopodia are intrinsically generated sensing structures. Therefore, nanoscale surveillance of brain parenchyma by microglia requires localized cAMP increases that drive filopodia formation. Shifting intracellular cAMP levels controls the polarity of microglial responses to changes in brain homeostasis and alters the scale of immunosurveillance.

First Extensive Microscopic Study of Butternut Defense Mechanisms Following Inoculation with the Canker Pathogen Ophiognomonia clavigignenti-juglandacearum Reveals Compartmentalization of Tissue Damage.

Ophiognomonia clavigignenti-juglandacearum endangers the survival of butternut (Juglans cinerea) throughout its native range. While screening for disease resistance, we found that artificial inoculations of 48 butternut seedlings with O. clavigignenti-juglandacearum induced the expression of external symptoms, but only after a period of dormancy. Before dormancy, compartmentalized tissues such as necrophylactic periderms (NPs) and xylem reaction zones (RZs) contributed to limiting pathogen invasion. Phenols were regularly detected in RZs, often in continuity with NPs during wound closure, and confocal microscopy revealed their presence in parenchyma cells, vessel plugs and cell walls. Vessels were blocked with tyloses and gels, particularly those present in RZs. Suberin was also detected in cells formed over the affected xylem by the callus at the inoculation point, in a few tylosis walls, and in longitudinal tubes that formed near NPs. Following dormancy, in all inoculated seedlings but one, defensive barriers were breached by O. clavigignenti-juglandacearum and then additional ones were produced in response to this new invasion. The results of this histopathological study indicate that trees inoculated in selection programs to test butternut canker resistance should go through at least one period of dormancy and that asymptomatic individuals should be dissected to better assess how they defend themselves against O. clavigignenti-juglandacearum.

CaV 3.2 drives sustained burst-firing, which is critical for absence seizure propagation in reticular thalamic neurons.

OBJECTIVE: Genetic alterations have been identified in the CACNA1H gene, encoding the CaV 3.2 T-type calcium channel in patients with absence epilepsy, yet the precise mechanisms relating to seizure propagation and spike-wave-discharge (SWD) pacemaking remain unknown. Neurons of the thalamic reticular nucleus (TRN) express high levels of CaV 3.2 calcium channels, and we investigated whether a gain-of-function mutation in the Cacna1h gene in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) contributes to seizure propagation and pacemaking in the TRN. METHODS: Pathophysiological contributions of CaV 3.2 calcium channels to burst firing and absence seizures were assessed in vitro using acute brain slice electrophysiology and quantitative real-time polymerase chain reaction (PCR) and in vivo using free-moving electrocorticography recordings. RESULTS: TRN neurons from GAERS display sustained oscillatory burst-firing that is both age- and frequency-dependent, occurring only in the frequencies overlapping with GAERS SWDs and correlating with the expression of a CaV 3.2 mutation-sensitive splice variant. In vivo knock-down of CaV 3.2 using direct thalamic injection of lipid nanoparticles containing CaV 3.2 dicer small interfering (Dsi) RNA normalized TRN burst-firing, and in free-moving GAERS significantly shortened seizures. SIGNIFICANCE: This supports a role for TRN CaV 3.2 T-type channels in propagating thalamocortical network seizures and setting the pacemaking frequency of SWDs.

3DMorph Automatic Analysis of Microglial Morphology in Three Dimensions from Ex Vivo and In Vivo Imaging.

Microglia are dynamic immune cells of the central nervous system, and their morphology is commonly used as a readout of cellular function. However, current morphological analysis techniques rely on either tracing of cells or two-dimensional projection analysis, which are time-consuming, subject to bias, and may ignore important three-dimensional (3D) information. Therefore, we have created 3DMorph, a MATLAB-based script that analyzes microglial morphology from 3D data. The program initially requires input of threshold levels, cell size expectations, and preferred methods of skeletonization. This makes 3DMorph easily scalable and adaptable to different imaging parameters or cell types. After these settings are defined, the program is completely automatic and can batch process files without user input. Output data includes cell volume, territorial volume, branch length, number of endpoints and branch points, and average distance between cells. We show that 3DMorph is accurate compared to manual tracing, with significantly decreased user input time. Importantly, 3DMorph is capable of processing in vivo microglial morphology, as well as other 3D branching cell types, from mouse cranial windows or acute hippocampal slices. Therefore, we present a novel, user-friendly, scalable, and semiautomatic method of analyzing cell morphology in 3 dimensions. This method should improve the accuracy of cell measurements, remove user bias between conditions, increase reproducibility between experimenters and labs, and reduce user input time. We provide this open source code on GitHub so that it is free and accessible to all investigators.