Cell Axons Research Articles

A high-efficiency model indicating the role of inhibition in the resilience of neuronal networks to damage resulting from traumatic injury.

Recent investigations of traumatic brain injuries have shown that these injuries can result in conformational changes at the level of individual neurons in the cerebral cortex. Focal axonal swelling is one consequence of such injuries and leads to a variable width along the cell axon. Simulations of the electrical properties of axons impacted in such a way show that this damage may have a nonlinear deleterious effect on spike-encoded signal transmission. The computational cost of these simulations complicates the investigation of the effects of such damage at a network level. We have developed an efficient algorithm that faithfully reproduces the spike train filtering properties seen in physical simulations. We use this algorithm to explore the impact of focal axonal swelling on small networks of integrate and fire neurons. We explore also the effects of architecture modifications to networks impacted in this manner. In all tested networks, our results indicate that the addition of presynaptic inhibitory neurons either increases or leaves unchanged the fidelity, in terms of bandwidth, of the network's processing properties with respect to this damage.

Fibrillin-1 mutant mouse captures defining features of human primary open glaucoma including anomalous aqueous humor TGF beta-2

Primary open angle glaucoma (POAG) features an optic neuropathy, elevated aqueous humor (AH) TGFβ2, and major risk factors of central corneal thickness (CCT), increasing age and intraocular pressure (IOP). We examined Tight skin (Tsk) mice to see if mutation of fibrillin-1, a repository for latent TGFβ, is associated with characteristics of human POAG. We measured: CCT by ocular coherence tomography (OCT); IOP; retinal ganglion cell (RGC) and optic nerve axon counts by microscopic techniques; visual electrophysiologic scotopic threshold responses (STR) and pattern electroretinogram (PERG); and AH TGFβ2 levels and activity by ELISA and MINK epithelial cell-based assays respectively. Tsk mice had open anterior chamber angles and compared with age-matched wild type (WT) mice: 23% thinner CCT (p < 0.003); IOP that was higher (p < 0.0001), more asymmetric (p = 0.047), rose with age (p = 0.04) and had a POAG-like frequency distribution. Tsk mice also had RGCs that were fewer (p < 0.04), declined with age (p = 0.0003) and showed increased apoptosis and glial activity; fewer optic nerve axons (p = 0.02); abnormal axons and glia; reduced STR (p < 0.002) and PERG (p < 0.007) visual responses; and higher AH TGFβ2 levels (p = 0.0002) and activity (p = 1E−11) especially with age. Tsk mice showed defining features of POAG, implicating aberrant fibrillin-1 homeostasis as a pathogenic contributor to emergence of a POAG phenotype.

Исследование решетчатой мембраны склеры при глаукоме

The main damage in glaucoma starts at the level of the sclera, where the axons of the retinal ganglion cells forming the optic nerve fibers and retinal vessels pass through a network of connective tissue called the lamina cribrosa (LC). Modern diagnostic methods, such as optical coherence tomography, especially SD-OCT and Swept-OCT (SS-OCT), make it possible to visualize LC and determine blood flow in it, opening up new possibilities for the diagnosis of glaucoma. The review provides information about the anatomy and blood supply of the LC, as well as the latest data on the study of these structures by optical coherence tomography. Key words: primary open-angle glaucoma, lamina cribrosa, ocular blood flow, swept-source optical coherence tomography, pressure of cerebrospinal fluid

Modeling neuron growth using isogeometric collocation based phase field method

We present a new computational framework of neuron growth based on the phase field method and develop an open-source software package called “NeuronGrowth_IGAcollocation”. Neurons consist of a cell body, dendrites, and axons. Axons and dendrites are long processes extending from the cell body and enabling information transfer to and from other neurons. There is high variation in neuron morphology based on their location and function, thus increasing the complexity in mathematical modeling of neuron growth. In this paper, we propose a novel phase field model with isogeometric collocation to simulate different stages of neuron growth by considering the effect of tubulin. The stages modeled include lamellipodia formation, initial neurite outgrowth, axon differentiation, and dendrite formation considering the effect of intracellular transport of tubulin on neurite outgrowth. Through comparison with experimental observations, we can demonstrate qualitatively and quantitatively similar reproduction of neuron morphologies at different stages of growth and allow extension towards the formation of neurite networks.

Mechanisms Underlying Mu Opioid Receptor Effects on Parallel Fiber-Purkinje Cell Synaptic Transmission in Mouse Cerebellar Cortex.

μ-opioid receptors (MOR) are widely expressed in the brain, varying in density in different areas. Activation of MORs underlies analgesia, euphoria, but may lead to tolerance, dependence, and ultimately opioid addiction. The Purkinje cell (PC) is the only efferent neuron in the cerebellar cortex and receives glutamatergic synaptic inputs from the parallel fibers formed by the axons of granule cells. Studies have shown that MORs are expressed during the development of cerebellar cells. However, the distribution of MOR and their effects on PF-PC synaptic transmission remain unclear. To examine these questions, we used whole-cell patch clamp recordings and pharmacological methods to determine the effects and mechanisms of MOR activation on synaptic transmission at PF-PC synapses. The MOR-selective agonist DAMGO significantly reduced the amplitude and area under the curve (AUC) of PF-PC evoked (e) EPSCs, and increased the paired-pulse ratio (PPR).DAMGO-induced inhibitory effects on PF-PC eEPSCs and PPR were abolished by MOR specific blocker CTOP. Further, DAMGO significantly reduced the frequency of PF-PC mEPSCs, but had no obvious effect on their amplitude, suggesting a presynaptic site of action. The DAMGO-induced reduction in the frequency of PF-PC mEPSCs also was blocked by CTOP. A protein kinase A (PKA) inhibitor PKI added in the pipette solution did not affect the inhibitory effects on PF-PC mEPSCs induced by DAMGO. Both the PKA inhibitor K5720 and MEK inhibitor U0126 in artificial cerebrospinal fluid (ACSF) prevented the inhibitory effects of DAMGO on PF-PC mEPSCs. These findings reveal that MORs are expressed in presynaptic PF axon terminals, where DAMGO can activate presynaptic MORs to inhibit PF-PC synaptic transmission by regulating the release of glutamate. G-protein-dependent cAMP-PKA signaling pathway may be involved in this process.

Transcriptional control of retinal ganglion cell death after axonal injury

Injury to the axons of retinal ganglion cells (RGCs) is a key pathological event in glaucomatous neurodegeneration. The transcription factors JUN (the target of the c-Jun N-terminal kinases, JNKs) and DDIT3/CHOP (a mediator of the endoplasmic reticulum stress response) have been shown to control the majority of proapoptotic signaling after mechanical axonal injury in RGCs and in other models of neurodegeneration. The downstream transcriptional networks controlled by JUN and DDIT3, which are critical for RGC death, however, are not well defined. To determine these networks, RNA was isolated from the retinas of wild-type mice and mice deficient in Jun, Ddit3, and both Jun and Ddit3 three days after mechanical optic nerve crush injury (CONC). RNA-sequencing data analysis was performed and immunohistochemistry was used to validate potential transcriptional signaling changes after axonal injury. This study identified downstream transcriptional changes after injury including both neuronal survival and proinflammatory signaling that were attenuated to differing degrees by loss of Ddit3, Jun, and Ddit3/Jun. These data suggest proinflammatory signaling in the retina might be secondary to activation of pro-death pathways in RGCs after acute axonal injury. These results determine the downstream transcriptional networks important for apoptotic signaling which may be important for ordering and staging the pro-degenerative signals after mechanical axonal injury.

An ESCRT/VPS4 Envelopment Trap To Examine the Mechanism of Alphaherpesvirus Assembly and Transport in Neurons.

The assembly and egress of alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), within neurons is poorly understood. A key unresolved question is the structure of the viral particle that moves by anterograde transport along the axon, and two alternative mechanisms have been described. In the "married" model, capsids acquire their envelopes in the cell body and then traffic along axons as enveloped virions within a bounding organelle. In the "separate" model, nonenveloped capsids travel from the cell body into and along the axon, eventually encountering their envelopment organelles at a distal site, such as the nerve cell terminal. Here, we describe an "envelopment trap" to test these models using the dominant negative terminal endosomal sorting complex required for transport (ESCRT) component VPS4-EQ. Green fluorescent protein (GFP)-tagged VPS4-EQ was used to arrest HSV-1 or PRV capsid envelopment, inhibit downstream trafficking, and GFP-label envelopment intermediates. We found that GFP-VPS4-EQ inhibited trafficking of HSV-1 capsids into and along the neurites and axons of mouse CAD cells and rat embryonic primary cortical neurons, consistent with egress via the married pathway. In contrast, transport of HSV-1 capsids was unaffected in the neurites of human SK-N-SH neuroblastoma cells, consistent with the separate mechanism. Unexpectedly, PRV (generally thought to utilize the married pathway) also appeared to employ the separate mechanism in SK-N-SH cells. We propose that apparent differences in the methods of HSV-1 and PRV egress are more likely a reflection of the host neuron in which transport is studied rather than true biological differences between the viruses themselves. IMPORTANCE Alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), are pathogens of the nervous system. They replicate in the nerve cell body and then travel great distances along axons to reach nerve termini and spread to adjacent epithelial cells; however, key aspects of how these viruses travel along axons remain controversial. Here, we test two alternative mechanisms for transport, the married and separate models, by blocking envelope assembly, a critical step in viral egress. When we arrest formation of the viral envelope using a mutated component of the cellular ESCRT apparatus, we find that entry of viral particles into axons is blocked in some types of neurons but not others. This approach allows us to determine whether envelope assembly occurs prior to entry of viruses into axons or afterwards and, thus, to distinguish between the alternative models for viral transport.

The exit of axons and glial membrane from the developing Drosophila retina requires integrins

Coordinated development of neurons and glia is essential for the establishment of neuronal circuits during embryonic development. In the developing Drosophila visual system, photoreceptor (R cell) axons and wrapping glial (WG) membrane extend from the eye disc through the optic stalk into the optic lobe. Extensive studies have identified a number of genes that control the establishment of R-cell axonal projection pattern in the optic lobe. The molecular mechanisms directing the exit of R-cell axons and WG membrane from the eye disc, however, remain unknown. In this study, we show that integrins are required in R cells for the extension of R-cell axons and WG membrane from the eye disc into the optic stalk. Knockdown of integrins in R cells but not WG caused the stalling of both R-cell axons and WG membrane in the eye disc. Interfering with the function of Rhea (i.e. the Drosophila ortholog of vertebrate talin and a key player of integrin-mediated adhesion), caused an identical stalling phenotype. These results support a key role for integrins on R-cell axons in directing R-cell axons and WG membrane to exit the eye disc.

Морфологія нюхового аналізатора людини. Лекція

The sense of smell provides people with valuable information about the biochemical environment and their own body. Olfactory disorders occur in pathologies of the nasal cavity, liver cirrhosis, psychological and endocrine diseases. Smell affects various psychological aspects of people's lives, forming positive and negative emotional memories associated with smells. With the dysfunction of the olfactory analyzer, a person will not do the analysis whether the food is good, will not be able to feel the presence of poisonous gases in the air, bad breath. This puts a person in an awkward position and increases the risk of social isolation. The purpose of the study was to highlight the components of the normal structure and functioning of the human olfactory analyzer. Identification of odors in the environment and from one's own body is provided by the olfactory analyzer. Primary odors as camphor, floral, fruity, spicy, tarry, burnt and putrid in different quantities form secondary odors. Aromas are composed of volatile molecules called odorants. The smallest amount of odorant that causes an odor sensation is called the odor threshold. In people with coronavirus disease the sense of smell temporarily disappears (anosmia); it is reduced (hyposmia) in liver cirrhosis and rhinitis, and in Alzheimer's disease and schizophrenia besides hyposmia there is olfactory hallucination (phantosmia). Olfactory dysfunction adversely affects children's cognitive abilities. Fragrances change emotions and behavior. Aromas are used to regulate the physical and psychological state of the patient. Volatile molecules of fragrances penetrate through the layer of mucus that covers the olfactory epithelium located in the olfactory region of the nasal mucosa. The olfactory epithelium consists of olfactory, supportive and basal epitheliocytes, as well as secretory cells of the olfactory glands. Olfactory cells are modified nerve cells that have a body, an axon, and a dendrite, which ends with a receptor in the form of olfactory cilia. Volatile molecules interact with the olfactory cilia and then with the receptor protein, which is located on the olfactory cell bodies. In humans, olfactory cells have 350 receptor proteins. One type of receptor can register molecules of several different odorants. Molecules of the same odorant can activate several different receptors simultaneously. The nerve impulse from the olfactory cells (bodies of I neurons) reaches the nerve cells (bodies of II neurons) of the olfactory bulbs via their central outgrowths (olfactory filaments). Axons of nerve cells of olfactory bulbs continue to bodies of III neurons, which are located in subcortical centers of the brain (almond-shaped body, nuclei of the transparent septum). In human, to analyze a particular odor, axons from bodies of III neurons continue to cortex, namely to the area of the uncus of the parahippocampal gyrus

A model of lateral interactions as the origin of multiwhisker receptive fields in rat barrel cortex.

While cells within barrel cortex respond primarily to deflections of their principal whisker (PW), they also exhibit responses to non-principal, or adjacent, whiskers (AWs), albeit responses with diminished amplitudes and longer latencies. The origin of multiwhisker receptive fields of barrel cells remains a point of controversy within the experimental literature, with three contending possibilities: (i) barrel cells inherit their AW responses from the AW responses of thalamocortical (TC) cells within their aligned barreloid; (ii) the axons of TC cells within a barreloid ramify to innervate multiple barrels, rather than only terminating within their aligned barrel; (iii) lateral intracortical transmission between barrels conveys AW responsivity to barrel cells. In this work, we develop a detailed, biologically plausible model of multiple barrels in order to examine possibility (iii); in order to isolate the dynamics that possibility (iii) entails, we incorporate lateral connections between barrels while assuming that TC cells respond only to their PW and that TC cell axons are confined to their home barrel. We show that our model is capable of capturing a broad swath of experimental observations on multiwhisker receptive field dynamics within barrels, and we compare and contrast the dynamics of this model with model dynamics from prior work in which employ a similar general modeling strategy to examine possibility (i).

Enhancing Cohort PASA Efficiency from Lessons Assimilated by Mutant Genotyping in C. elegans.

Classical restriction fragment length polymorphism (RFLP) and sequencing are labor-intensive and expensive methods to study single base changes, whereas polymerase chain reaction amplification of specific alleles (PASA) or allele-specific polymerase chain reaction (ASPCR) is a PCR-based application that allows direct detection of any point mutation by analyzing the PCR products in an ethidium bromide-stained agarose or polyacrylamide gel. PASA is based on oligonucleotide primers containing one or more 3' mismatch with the target DNA making it refractory to primer extension by Thermus aquaticus DNA polymerase lacking the 3' to 5' exonuclease proofreading activity because of which it is also called amplification refractory mutation system-PCR (ARMS-PCR). This technique has found application in detection of allele, mutation, single-nucleotide polymorphisms (SNPs) causing genetic and infectious diseases. This chapter describes an approach of cohort PASA in context of genotyping single and double mutant worms generated to study the process of cell migration and axon outgrowth in C. elegans. Single worm-based cohort PASA allows genotyping for identification of single base mutations; particularly it is convenient method to detect mutations without a visible phenotype.

CDKL5 Gene: Beyond Rett Syndrome

AbstractCDKL5 is a gene located in the X-chromosome (Xp22) encoding a serine/threonine kinase involved in various signaling pathways, implicated in cell proliferation, axon development, dendrite growth, synapse formation, and maintenance. Mutations occurring in this gene have been associated with drug-resistant early-onset epilepsy, with multiple seizures type, and deep cognitive and motor development delay with poor or absent speech, ataxic gait or inability to walk, hand stereotypies and in a few cases decrement of head growth. Many aspects remain unclear about the CDKL5 deficiency disorders, research will be fundamental to better understand the pathogenesis of neurological damage and consequently developed more targeted and profitable therapies, as there is not, at the present, a gene-based treatment and the seizures are in most of the cases drug resistant. In this article, we summarize the actual knowledge about CDKL5 gene function and mostly the consequence given by its dysfunction, also examining the possible therapeutic approaches.

Betacellulin regulates peripheral nerve regeneration by affecting Schwann cell migration and axon elongation

BackgroundGrowth factors execute essential biological functions and affect various physiological and pathological processes, including peripheral nerve repair and regeneration. Our previous sequencing data showed that the mRNA coding for betacellulin (Btc), an epidermal growth factor protein family member, was up-regulated in rat sciatic nerve segment after nerve injury, implying the potential involvement of Btc during peripheral nerve regeneration.MethodsExpression of Btc was examined in Schwann cells by immunostaining. The function of Btc in regulating Schwann cells was investigated by transfecting cultured cells with siRNA segment against Btc or treating cells with Btc recombinant protein. The influence of Schwann cell-secreted Btc on neurons was determined using a co-culture assay. The in vivo effects of Btc on Schwann cell migration and axon elongation after rat sciatic nerve injury were further evaluated.ResultsImmunostaining images and ELISA outcomes indicated that Btc was present in and secreted by Schwann cells. Transwell migration and wound healing observations showed that transfection with siRNA against Btc impeded Schwann cell migration while application of exogenous Btc advanced Schwann cell migration. Besides the regulating effect on Schwann cell phenotype, Btc secreted by Schwann cells influenced neuron behavior and increased neurite length. In vivo evidence supported the promoting role of Btc in nerve regeneration after both rat sciatic nerve crush injury and transection injury.ConclusionOur findings demonstrate the essential roles of Btc on Schwann cell migration and axon elongation and imply the potential application of Btc as a regenerative strategy for treating peripheral nerve injury.

Light and Transmission Electron Microscopic Study of Olfactory Organ of Spotted Snakehead, Channa punctata (Bloch)

The olfactory organ of Channa punctata (Bloch) has been studied under light microscope and transmission electron microscope. The olfactory apparatus of C. punctata comprises of olfactory rosette, olfactory nerve, and olfactory bulb. Paired, oval shaped olfactory rosette present in the olfactory chamber on fish rostrum. Olfactory chamber opens externally via an anterior inlet and a posterior nostril outlet. Olfactory epithelium comprises of sensory and non-sensory regions. Sensory region is at basal part and non-sensory region is at proximal area of olfactory lamellae. In olfactory lamellae, sensory epithelium consists of olfactory receptor cells, supporting cells, basal cells, goblet cells, mucous cell and white cell. Olfactory receptor cells are of two types microvillous and ciliated. In C. punctata, olfactory bulb is sessile and is attached to the telencephalon. Cells of olfactory bulb are organized in four concentric layers. Outer layer is olfactory nerve layer formed by axons of olfactory receptor cells. Inner to it is glomerular layer where axons of olfactory receptor cells synapse with dendrites of mitral cells. Next towards deeper part is mitral cell layer which comprises larger multipolar mitral cells. Central core is formed by granular cell layer with small granular cells.

Selective effects of protein 4.1N deficiency on neuroendocrine and reproductive systems

Protein 4.1N, a member of the protein 4.1 family, is highly expressed in the brain. But its function remains to be fully defined. Using 4.1N−/− mice, we explored the function of 4.1N in vivo. We show that 4.1N−/− mice were born at a significantly reduced Mendelian ratio and exhibited high mortality between 3 to 5 weeks of age. Live 4.1N−/− mice were smaller than 4.1N+/+ mice. Notably, while there were no significant differences in organ/body weight ratio for most of the organs, the testis/body and ovary/body ratio were dramatically decreased in 4.1N−/− mice, demonstrating selective effects of 4.1N deficiency on the development of the reproductive systems. Histopathology of the reproductive organs showed atrophy of both testis and ovary. Specifically, in the testis there is a lack of spermatogenesis, lack of leydig cells and lack of mature sperm. Similarly, in the ovary there is a lack of follicular development and lack of corpora lutea formation, as well as lack of secretory changes in the endometrium. Examination of pituitary glands revealed that the secretory granules were significantly decreased in pituitary glands of 4.1N−/− compared to 4.1N+/+. Moreover, while GnRH was expressed in both neuronal cell body and axons in the hypothalamus of 4.1N+/+ mice, it was only expressed in the cell body but not the axons of 4.1N-/- mice. Our findings uncover a novel role for 4.1N in the axis of hypothalamus-pituitary gland-reproductive system.

Topical Ocular Delivery of Nanocarriers: A Feasible Choice for Glaucoma Management.

Topical ocular delivery is an acceptable and familiar approach for the treatment of common ocular diseases. Novel strategies for the treatment of inherited eye diseases include new pharmacologic agents, gene therapy and genome editing, which lead to the expansion of new management options for eye disorders. The topical ocular delivery of nanocarriers is a technique, which has the potential to facilitate novel treatments. Nanocarrier- based strategies have proven effective for site-targeted delivery. This review summarizes recent development in the area of topical delivery of different nanocarriers (Polymer, Vesicular and dispersed systems) for the management of glaucoma, a group of ocular disorders characterized by progressive and accelerated degeneration of the axons of retinal ganglion cells, which make up the optic nerve. Unique cellular targets for glaucoma treatment, primarily the trabecular meshwork of the anterior segment of the eye, make glaucoma facilitated by the use of nanocarriers an ideal disorder for novel molecular therapies.

Nusap1, Melk and L1cam Expression in Cervical Cancer Tissues; Prognostic and Clinicopathological Implications

Background: It is important to identify of novel therapeutic targets for cervical carcinoma. Nucleolar and spindle associated protein 1 (NUSAP1) could be able to bind microtubules, and has important role in spindle formation and mitotic progression. Maternal embryonic leucine zipper kinase (MELK) is a cell cycle related conserved protein kinase that is involved in control and regulation of the cell cycle, apoptosis, and carcinogenesis. The L1 cell adhesion molecule (L1-CAM) was discovered as a protein which has an essential role in cell migration and axon guidance in the nervous system. The Aim of This Study Is to investigate the tissue protein expression of NUSAP1, MELK and L1CAM in tissues of cervical carcinoma and to detect their prognostic roles. Methods: Expression of NUSAP1, MELK and L1CAM was evaluated in sections from 62 cases of cervical carcinoma using immunohistochemistry we followed our patients for 3 years correlate their expression with clinicopathological parameters and patients outcome. Results: High levels of NUSAP1, MELK and L1CAM expression was positively related to older age of the patients, high grade of the tumor, lympho-vascular invasion, advanced FIGO stage, poor survival rates and higher recurrence rate after successful therapy (p&lt;0.001). L.N metastases (p=0.002), distant metastases (p=0.003). There are statistically significant positive association between NUSAP1, MELK and L1CAM Phi correlation coefficient= + 0.87 and + 0.721 respectively (p&lt;0.001). Conclusion: NUSAP1, MELK and L1CAM are considered markers of poor prognosis in cervical carcinoma cells.

ЗАКОНОМІРНОСТІ СПОНТАННОГО КВАНТОВОГО ВИВІЛЬНЕННЯ ГЛУТАМАТУ В СИНАПСАХ НЕЙРОНІВ ПЕРВИННОЇ КУЛЬТУРИ ГІПОКАМПА ПРИ ДОВГОТРИВАЛІЙ ДЕПРЕСІЇ СИНАПТИЧНОЇ ПЕРЕДАЧІ

The results of electrophysiological studies of the quantal release features of glutamate in hippocampal neurons' synapses in long-term depression of synaptic transmission are presented. To research the topic, weused the primary culture of the rat hippocampus. Using the patch-clamp technique, the frequencies and amplitudes of excitatory spontaneous postsynaptic currents (EPSCs) were determined, theirdistributions were analyzed and the basicquantal parameters were calculated. Long-term depression of synaptic transmission was achieved by tetanic stimulation of the axon of the presynaptic cell for 5 minutes with a stimulation frequency of 5 Hz. Spontaneous current in depression was recorded 20-30 min after tetanic stimulation. It has been shown that the amplitude of the EPSCs registered in hippocampal neurons decreased in the long-term depression of synaptic transmission. It has been found that two neurotransmitter vesicles are usually released in the control at the same time and one during the long-term depression of synaptic transmission.It means that the probability of release decreased. In addition, the average quantalamplitude and quantum content decreased. It indicates the effect of presynaptic mechanisms in the expression of long-term depression of synaptic neurotransmission between hippocampal neurons in culture.In the conditions of long-term depression, no significant changes were found in the frequency of EPSCs. The analysis of quantum parameters in long-term depression of glutamatergic synaptic transmission between neurons is important for the formation of more complete ideas about the mechanisms that play a fundamental role in the normal functioning of the CNS and the development of neural networks.

Preparation of Herpes Simplex Virus-Infected Primary Neurons for Transmission Electron Microscopy.

Transmission electron microscopy (TEM) provides the resolution necessary to identify both viruses and subcellular components of cells infected with many types of viruses, including herpes simplex virus. Recognized as a powerful tool in both diagnostic and research-based virology laboratories, TEM has made possible the identification of new viruses and has contributed to the elucidation of virus life cycle and virus-host cell interaction.While there are many sample preparation techniques for TEM, conventional processing using chemical fixation and resin embedding remains a useful technique, available in virtually all EM laboratories, for studying virus/cell ultrastructure. In this chapter, we describe the preparation of herpes simplex virus infected primary neurons, grown on plastic coverslips, to allow for sectioning of neurons and axons in their growth plane. This technique allows for TEM examination of cell bodies, axons, growth cones and varicosities, providing powerful insights into virus-cell interaction.

The Use of Microfluidic Neuronal Devices to Study the Anterograde Axonal Transport of Herpes Simplex Virus-1.

Understanding how herpes simplex virus-1 (HSV-1) interacts with different parts of the neuron is fundamental in understanding the mechanisms behind HSV-1 transport during primary and recurrent infections. In this chapter, we describe a unique neuronal culture system that is capable of compartmentalizing neuronal cell bodies from their axons to study the transport of HSV-1 along axons. The ability to separate neuronal cell bodies and axons provides a unique model to investigate the mechanisms used by HSV-1 for viral transport, assembly, and exit from different parts of the neuron.