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Representative examples of triple immunofluorescence staining of mouse brain tissue using 1% formaldehyde and 0.5% MeOH fixation. (A–D) For the entire figure, the gray and red channels represent FGF14 immunoreactivity, the green NeuN, and blue Ankyrin-G (NeuroMab, catalog number 75-146) in indicated brain regions. The corresponding multichannel overlaid images are shown in the right column. DG, dentate gyrus; NeuN, Neuronal marker. Scale bars represent 20 μm.
Source publication
The axonal initial segment (AIS) is the subcellular compartment required for initiation of the action potential in neurons. Scaffolding and regulatory proteins at the AIS cluster with ion channels ensuring the integrity of electrical signaling. Interference with the configuration of this protein network can lead to profound effects on neuronal pola...
Citations
... Fifteen micrometer slices were prepared from brain tissue previously frozen in liquid nitrogen vapors and then slide mounted and washed with 1× PBS for 5 min, followed by 7 min cold acetone fixation/permeabilization, as has been described previously for imaging AIS proteins (Alshammari et al., 2016;Di Re et al., 2019). Tissue was washed in 1× PBS (3 times, 10 min each), blocked using a 10% NGS solution (Life Technologies, Carlsbad, CA, United States, #50062Z) for 30 min, and stained using the following primary antibodies overnight at 4°C: anti-FGF13 msIgG2b (Invitrogen, Catalog # MA5-27705) and anti-AnkyrinG msIgG2a (Antibodies Incorporated, Davis, CA, United States, 75-146). ...
Cocaine use disorder (CUD) is a prevalent neuropsychiatric disorder with few existing treatments. Thus, there is an unmet need for the identification of new pharmacological targets for CUD. Previous studies using environmental enrichment versus isolation paradigms have found that the latter induces increased cocaine self-administration with correlative increases in the excitability of medium spiny neurons (MSN) of the nucleus accumbens shell (NAcSh). Expanding upon these findings, we sought in the present investigation to elucidate molecular determinants of these phenomena. To that end, we first employed a secondary transcriptomic analysis and found that cocaine self-administration differentially regulates mRNA for fibroblast growth factor 13 (FGF13), which codes for a prominent auxiliary protein of the voltage-gated Na⁺ (Nav) channel, in the NAcSh of environmentally enriched rats (i.e., resilient behavioral phenotype) compared to environmentally isolated rats (susceptible phenotype). Based upon this finding, we used in vivo genetic silencing to study the causal functional and behavioral consequences of knocking down FGF13 in the NAcSh. Functional studies revealed that knockdown of FGF13 in the NAcSh augmented excitability of MSNs by increasing the activity of Nav channels. These electrophysiological changes were concomitant with a decrease in cocaine demand elasticity (i.e., susceptible phenotype). Taken together, these data support FGF13 as being protective against cocaine self-administration, which positions it well as a pharmacological target for CUD.
... For the respective negative controls (mock), ten 1-day postnatal BALB/c mice (2 litters) were inoculated with a virusfree solution. When the mice showed advanced signs of infection (10 dpi), they were euthanized following the recommendations for immunohistochemical studies of the central nervous system (CNS) [65,66], which consists of fixing the brains via intracardiac perfusion. ...
Zika virus (ZIKV) disease continues to be a threat to public health, and it is estimated that millions of people have been infected and that there have been more cases of serious complications than those already reported. Despite many studies on the pathogenesis of ZIKV, several of the genes involved in the malformations associated with viral infection are still unknown. In this work, the morphological and molecular changes in the cortex and cerebellum of mice infected with ZIKV were evaluated. Neonatal BALB/c mice were inoculated with ZIKV intraperitoneally, and the respective controls were inoculated with a solution devoid of the virus. At day 10 postinoculation, the mice were euthanized to measure the expression of the markers involved in cortical and cerebellar neurodevelopment. The infected mice presented morphological changes accompanied by calcifications, as well as a decrease in most of the markers evaluated in the cortex and cerebellum. The modifications found could be predictive of astrocytosis, dendritic pathology, alterations in the regulation systems of neuronal excitation and inhibition, and premature maturation, conditions previously described in other models of ZIKV infection and microcephaly.
... The whole brain was removed and kept in 4% paraformaldehyde; 24 hours later, the solution was changed to 1% paraformaldehyde and maintained until cutting. Previous to cutting, brains were cryopreserved in 0.1 M PBS containing 30% sucrose (Bio-Lab ltd, Israel) for 48 h [32]. Two hours before cutting, brains were drained, dried, and frozen at −4 ºC. ...
Background
In the fear memory network, the hippocampus modulates contextual aspects of fear learning while mutual connections between the amygdala and the medial prefrontal cortex are widely involved in fear extinction. G-protein-coupled receptors (GPCRs) are involved in the regulation of fear and anxiety, so the regulation of GPCRs in fear signaling pathways can modulate the mechanisms of fear memory acquisition, consolidation and extinction. Various studies suggested a role of M-type K+ channels in modulating fear expression and extinction, although conflicting data prevented drawing of clear conclusions. In the present work, we examined the impact of M-type K+ channel blockade or activation on contextual fear acquisition and extinction. In addition, regarding the pivotal role of the hippocampus in contextual fear conditioning (CFC) and the involvement of the axon initial segment (AIS) in neuronal plasticity, we investigated whether structural alterations of the AIS in hippocampal neurons occurred during contextual fear memory acquisition and short-time extinction in mice in a behaviorally relevant context.
Results
When a single systemic injection of the M-channel blocker XE991 (2 mg/kg, IP) was carried out 15 minutes before the foot shock session, fear expression was significantly reduced. Expression of c-Fos was increased following CFC, mostly in GABAergic neurons at day 1 and day 2 post-fear training in CA1 and dentate gyrus hippocampal regions. A significantly longer AIS segment was observed in GABAergic neurons of the CA1 hippocampal region at day 2.
Conclusions
Our results underscore the role of M-type K + channels in CFC and the importance of hippocampal GABAergic neurons in fear expression.
... The axon initial segment (AIS) is the site of a neuron that separates its somatodendritic and axonal compartments, and is primarily responsible for maintaining the neuron's polarity and initiating action potentials . One of the most essential components of the AIS is the cytoskeletal-associated protein, Ankyrin-G (AnkG) (Alshammari et al., 2016). Previous studies have shown that shorter or fewer AISes measured by AnkG are indicative of impairments to the neuron's physiology, such as a decrease in excitability Galliano et al., 2021;Yamada and Kuba, 2016). ...
... AISes are responsible for action potential initiation and maintenance of neuronal polarity Hedstrom et al., 2008). The actual assembly of the AIS is coordinated primarily by AnkG, a cytoskeletal-associated protein (Alshammari et al., 2016;Jenkins and Bennett, 2001). It remains unknown whether the mechanisms of AIS maintenance and AIS assembly (or reassembly) as controlled by AnkG are related (Le Bras et al., 2014). ...
Chronic olfactory inflammation (COI) in conditions such as chronic rhinosinusitis significantly impairs the functional and anatomical components of the olfactory system. COI induced by intranasal administration of lipopolysaccharide (LPS) results in atrophy, gliosis, and pro-inflammatory cytokine production in the olfactory bulb (OB). Although chronic rhinosinusitis patients have smaller OBs, the consequences of olfactory inflammation on OB neurons are largely unknown. In this study, we investigated the neurological consequences of COI on OB projection neurons, mitral cells (MCs) and tufted cells (TCs). To induce COI, we performed unilateral intranasal administration of LPS to mice for 4 and 10 weeks. Effects of COI on the OB were examined using RNA-sequencing approaches and immunohistochemical analyses. We found that repeated LPS administration upregulated immune-related biological pathways in the OB after 4 weeks. We also determined that the length of TC lateral dendrites in the OB significantly decreased after 10 weeks of COI. The axon initial segment of TCs decreased in number and in length after 10 weeks of COI. The lateral dendrites and axon initial segments of MCs, however, were largely unaffected. In addition, dendritic arborization and AIS reconstruction both took place following a 10-week recovery period. Our findings suggest that olfactory inflammation specifically affects TCs and their integrated circuitry, whereas MCs are potentially protected from this condition. This data demonstrates unique characteristics of the OBs ability to undergo neuroplastic changes in response to stress.
... Following sectioning described above, rostro-caudally matched 300 µ m slices of either hemisphere were treated with 20 µ M triciribine (SelleckChem S1117) or 0.02% DMSO for 2 h in order to maximize the physiological changes that could be captured with immunohistochemistry and confocal imaging. Slices were then washed with 1× PBS for 5 min, fixed with 1% PFA with methanol (Sigma-Aldrich, St. Louis, MO, USA, #252549) for 30 min, and dehydrated in 20% sucrose overnight, as described previously [83]. The tissue was then embedded in OCT and sectioned at 30 µ m and mounted onto SuperFrost Plus slides (Fisher Scientific 12-550-15). ...
In neurons, changes in Akt activity have been detected in response to the stimulation of transmembrane receptors. However, the mechanisms that lead to changes in neuronal function upon Akt inhibition are still poorly understood. In the present study, we interrogate how Akt inhibition could affect the activity of the neuronal Nav channels with while impacting intrinsic excitability. To that end, we employed voltage-clamp electrophysiological recordings in heterologous cells expressing the Nav1.6 channel isoform and in hippocampal CA1 pyramidal neurons in the presence of triciribine, an inhibitor of Akt. We showed that in both systems, Akt inhibition resulted in a potentiation of peak transient Na+ current (INa) density. Akt inhibition correspondingly led to an increase in the action potential firing of the CA1 pyramidal neurons that was accompanied by a decrease in the action potential current threshold. Complementary confocal analysis in the CA1 pyramidal neurons showed that the inhibition of Akt is associated with the lengthening of Nav1.6 fluorescent intensity along the axonal initial segment (AIS), providing a mechanism for augmented neuronal excitability. Taken together, these findings provide evidence that Akt-mediated signal transduction might affect neuronal excitability in a Nav1.6-dependent manner.
... Na V 1.1 immunohistochemistry was performed as previously described (Alshammari et al., 2016). Briefly, mice were euthanized by isoflurane overdose and transcardially perfused with 1× PBS followed by ice-cold 1% PFA and 0.5% methanol in 1× PBS. ...
Thalamocortical network dysfunction contributes to seizures and sleep deficits in Dravet syndrome (DS), an infantile epileptic encephalopathy, but the underlying molecular and cellular mechanisms remain elusive. DS is primarily caused by mutations in the SCN1A gene encoding the voltage-gated sodium channel NaV1.1, which is highly expressed in GABAergic reticular thalamus (nRT) neurons as well as glutamatergic thalamocortical neurons. We hypothesized that NaV1.1 haploinsufficiency alters somatosensory corticothalamic circuit function through both intrinsic and synaptic mechanisms in nRT and thalamocortical neurons. Using Scn1a heterozygous mice of both sexes aged P25-P30, we discovered reduced excitability of nRT neurons and thalamocortical neurons in the ventral posterolateral (VPL) thalamus, while thalamocortical ventral posteromedial (VPM) neurons exhibited enhanced excitability. NaV1.1 haploinsufficiency enhanced GABAergic synaptic input and reduced glutamatergic input to VPL neurons, but not VPM neurons. In addition, glutamatergic input to nRT neurons was reduced in Scn1a heterozygous mice. These findings introduce alterations in glutamatergic synapse function and aberrant glutamatergic neuron excitability in the thalamus as disease mechanisms in DS, which has been widely considered a disease of GABAergic neurons. This work reveals additional complexity that expands current models of thalamic dysfunction in DS and identifies new components of corticothalamic circuitry as potential therapeutic targets.
... Несмотря на доведенную до предела разрешающую способность современных цифровых микроскопов, цветовую составляющую снимка практически не совершенствуют на аппаратном уровне. Основную работу с цветом производят уже после регистрации снимка или уделяется особое внимание фильтрации излучения для детальной работы с ультрафиолетовой областью спектра и флуоресценцией [1][2][3][4]. Разработка же представленная в данном труде позволяет работать в видимом диапазоне спектра. ...
The purpose of the work is to study the developed color separation systems based on the color triangle. Similar systems are planned to be used for matrix photodetectors of digital microscopes. Within the framework of this goal, the following tasks were formulated: development of color separation systems and their study. To solve the problems of developing a color separation system for a matrix photodetector, the main provisions of colorimetry, colorimetric systems, their transformations and methods for creating color spaces were used to determine the optimal color space with a color gamut of up to 100% of the visible color, the characteristics of which do not have negative branches, which will allow them to be implemented in the system color separation based on light filters. Based on the results of a mathematical study, a universal set with a GLC-3 system was selected with an average deviation from 14 nominal color values from the Munsell atlas of 0.0083. The selected space has a color gamut of 100% visible colors and an operating range of 400 nm to 730 nm. According to the results of the calculation, it can be assumed that the GLC-3 color space is the most suitable due to the similarity of the addition curves of this system with the curves of the CIEXYZ 1931 system, where the red channel curve is also slightly higher than the green one. Further, it is planned to introduce the developed color separation system into a digital microscope for further study of the obtained color images.
... Spinal cords were cut into 30-50 μm thick cross-sections using a Leica cryostat or microtome (Leica Microsystems, Buffalo Grove, IL) and mounted on glass microscope slides (Superfrost Plus,Fisher Scientific). Slides were incubated for 7-10 mins in cold acetone to reveal ankyrin-G (AnkG) epitopes [63]. Sections were then washed in 0.01M phosphate buffered saline with 0.3% triton (PBS-T) and incubated in blocking solution (10% normal goat serum mixed in PBS-T) for 1 hr. ...
The axon initial segment (AIS) responsible for action potential initiation is a dynamic structure that varies and changes together with neuronal excitability. Like other neuron types, alpha motoneurons in the mammalian spinal cord express heterogeneity and plasticity in AIS geometry, including length (AIS l) and distance from soma (AIS d). The present study aimed to establish the relationship of AIS geometry with a measure of intrinsic excitability, rheobase current, that varies by 20-fold or more among normal motoneurons. We began by determining whether AIS length or distance differed for motoneurons in motor pools that exhibit different activity profiles. Motoneurons sampled from the medial gastrocnemius (MG) motor pool exhibited values for average AIS d that were significantly greater than that for motoneurons from the soleus (SOL) motor pool, which is more readily recruited in low-level activities. Next, we tested whether AIS d covaried with intrinsic excitability of individual motoneurons. In anesthetized rats, we measured rheobase current intracellularly from MG motoneurons in vivo before labeling them for immunohistochemical study of AIS structure. For 16 motoneurons sampled from the MG motor pool, this combinatory approach revealed that AIS d , but not AIS l , was significantly related to rheobase, as AIS tended to be located further from the soma on motoneurons that were less excitable. Although a causal relation with excitability seems unlikely, AIS d falls among a constellation of properties related to the recruitability of motor units and their parent motoneurons.
... There exists severe cell shrinkage in ethanol fixation. PFA, as the most commonly used fixative for immunostaining of cells, has been associated with various problems, including loss of antigenicity and disruption of morphology during fixation [23,24]. ...
Mitochondria are highly dynamic organelles, constantly undergoing shape changes, which are controlled by mitochondrial movement, fusion, and fission. Mitochondria play a pivotal role in various cellular processes under physiological and pathological conditions, including metabolism, superoxide generation, calcium homeostasis, and apoptosis. Abnormal mitochondrial morphology and mitochondrial protein expression are always closely related to the health status of cells. Analysis of mitochondrial morphology and mitochondrial protein expression in situ is widely used to reflect the abnormality of cell function in the chemical fixed sample. Paraformaldehyde (PFA), the most commonly used fixative in cellular immunostaining, still has disadvantages, including loss of antigenicity and disruption of morphology during fixation. We tested the effect of ethanol (ETHO), PFA, and glutaraldehyde (GA) fixation on cellular mitochondria. The results showed that 3% PFA and 1.5% GA (PFA-GA) combination reserved mitochondrial morphology better than them alone in situ in cells. Mitochondrial network and protein antigenicity were well maintained, indicated by preserved MitoTracker and mitochondrial immunostaining after PFA-GA fixation. Our results suggest that the PFA-GA combination is a valuable fixative for the study of mitochondria in situ.
... To facilitate staining of Nav1.1 at the AES, we used very mild fixation (1% paraformaldehyde with 0.5% MeOH in PBS) described previously (Alshammari et al., 2016). Briefly, isoflurane-anesthetized mice were transcardially perfused; brains were removed and post-fixed in perfusate at RT for 1 hr. ...
GABAergic inhibitory interneurons of the cerebral cortex expressing vasoactive intestinal peptide (VIP-INs) are rapidly emerging as important regulators of network dynamics and normal circuit development. Several recent studies have also identified VIP-IN dysfunction in models of genetically determined neurodevelopmental disorders (NDDs). In this article, we review the known circuit functions of VIP-INs and how they may relate to accumulating evidence implicating VIP-INs in the mechanisms of prominent NDDs. We highlight recurring VIP-IN-mediated circuit motifs that are shared across cerebral cortical areas and how VIP-IN activity can shape sensory input, development, and behavior. Ultimately, we extract a set of themes that inform our understanding of how VIP-INs influence pathogenesis of NDDs. Using publicly available single-cell RNA sequencing data from the Allen Institute, we also identify several underexplored disease-associated genes that are highly expressed in VIP-INs. We survey these genes and their shared related disease phenotypes that may broadly implicate VIP-INs in autism spectrum disorder and intellectual disability rather than epileptic encephalopathy. Finally, we conclude with a discussion of the relevance of cell type-specific investigations and therapeutics in the age of genomic diagnosis and targeted therapeutics.
