Activation of microglia associated with lentiviral transduction: A semiautomated method of assessment.

Lentiviral transduction is a powerful tool and widely used in neuroscience research to manipulate gene expression of cells. However, the injection of lentiviral vectors in the brain is not totally benign, it potentially induces focal neuroinflammation. Upon inflammation, microglial cells get activated and can induce major changes in tissue environment, which may interfere with experimental results. In the current study, two weeks after the injection of control viral construction in the dentate gyrus (DG) of rats, an activation of microglia was detected. To access the activation status, we used a fast and accurate method of phenotype detection - measurement of fractal dimension (FD). Microglial morphology is a key indicator of neuroinflammation, therefore FD of microglial cells may serve as a reliable index of inflammation status in the brain. Here we present a detailed description of image processing procedure of images of individual microglial cells. The method allows to preserve the complex structure of microglial cells and their thin processes on the output image, which is important for accurate FD assessment.

Hippocampal expression of a virus-derived protein impairs memory in mice.

The analysis of the biology of neurotropic viruses, notably of their interference with cellular signaling, provides a useful tool to get further insight into the role of specific pathways in the control of behavioral functions. Here, we exploited the natural property of a viral protein identified as a major effector of behavioral disorders during infection. We used the phosphoprotein (P) of Borna disease virus, which acts as a decoy substrate for protein kinase C (PKC) when expressed in neurons and disrupts synaptic plasticity. By a lentiviral-based strategy, we directed the singled-out expression of P in the dentate gyrus of the hippocampus and we examined its impact on mouse behavior. Mice expressing the P protein displayed increased anxiety and impaired long-term memory in contextual and spatial memory tasks. Interestingly, these effects were dependent on P protein phosphorylation by PKC, as expression of a mutant form of P devoid of its PKC phosphorylation sites had no effect on these behaviors. We also revealed features of behavioral impairment induced by P protein expression but that were independent of its phosphorylation by PKC. Altogether, our findings provide insight into the behavioral correlates of viral infection, as well as into the impact of virus-mediated alterations of the PKC pathway on behavioral functions.

Cytomegalovirus in the human dentate gyrus and its impact on neural progenitor cells: report of two cases.

Congenital cytomegalovirus (CMV) infection in the 2nd and 3rd trimester results in catastrophic CNS abnormalities. This susceptibility is thought to result from the high proportion of neural stem cells in the developing brain. In immunocompromised adults, CNS infection by CMV preferentially affects ependymal surfaces, from where it expands to involve the parenchyma. Experimental models of murine CMV infection demonstrate viral tropism for the dentate gyrus (DG) and subventricular zone, the areas in which adult neurogenesis occurs. We present two cases of CMV infection of the DG of immunocompromised allogeneic stem cell transplant recipients. Both cases showed CMV-positive neurons in the DG granular cell layer, as well as contiguous layers. The majority of infected cells contained Nissl substance and expressed nestin, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), and neurofilament. These cases demonstrate that CMV infects the DG in humans. Co-expression of nestin and GFAP, indicative of early neurogenesis, is consistent with experimental models showing neural stem cells as the target of CMV, providing further histological evidence of neurogenesis in the human dentate. Finally, the cases suggest that CMV infection produces abnormal migration of newly formed neurons as evidenced by the finding of virally infected neurons in the molecular layer of the dentate.
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Lentiviral Transduction of Neurons in Adult Brain: Evaluation of Inflammatory Response and Cognitive Effects in Mice.

We evaluated the effect of hippocampal injection of lentiviral particles p156-CMV-EGFP on behavior, learning, and microglial Iba1(+) cells activation in mice. Testing in the open field and elevated plus-maze revealed higher anxiety levels in lentiviral-injected mice in comparison with animals injected with vehicle. At the same time, lentivirus injection did not change learning and memory of mice in the hippocampal-dependent fear conditioning task. Microglia density in lentivirus-injected mice was significantly higher than in vehicle-injected mice. Thus, hippocampal injection of lentiviral particles with minimum content of transgenes produced evident inflammation process, changed anxiety level of experimental animals, but had no effect on hippocampal-dependent learning and memory.

Induction of microglia activation after infection with the non-neurotropic A/CA/04/2009 H1N1 influenza virus.

Although influenza is primarily a respiratory disease, it has been shown, in some cases, to induce encephalitis, including people acutely infected with the pandemic A/California/04/2009 (CA/09) H1N1 virus. Based on previous studies showing that the highly pathogenic avian influenza (HPAI) A/Vietnam/1203/2004 H5N1 virus was neurotropic, induced CNS inflammation and a transient parkinsonism, we examined the neurotropic and inflammatory potential of the CA/09 H1N1 virus in mice. Following intranasal inoculation, we found no evidence for CA/09 H1N1 virus neurotropism in the enteric, peripheral or central nervous systems. We did, however, observe a robust increase in microglial activity in the brain characterized by an increase in the number of activated Iba-1-positive microglia in the substantia nigra (SN) and the hippocampus, despite the absence of virus in the brain. qPCR analysis in SN tissue showed that the induction of microgliosis was preceded by reduced gene expression of the neurotrophic factors bdnf, and gdnf and increases in the immune modulatory chemokine chemokine (C-C motif) ligand 4 (ccl4). We also noted changes in the expression of transforming growth factor-1 (tgfß1) in the SN starting at 7 days post-infection (dpi) that was sustained through 21 dpi, coupled with increases in arginase-1 (arg1) and csf1, M2 markers for microglia. Given that neuroinflammation contributes to generation and progression of a number of neurodegenerative disorders, these findings have significant implications as they highlight the possibility that influenza and perhaps other non-neurotropic viruses can initiate inflammatory signals via microglia activation in the brain and contribute to, but not necessarily be the primary cause of, neurodegenerative disorders.

Congenitally acquired persistent lymphocytic choriomeningitis viral infection reduces neuronal progenitor pools in the adult hippocampus and subventricular zone.

Lymphocytic choriomeningitis virus (LCMV) can be transmitted through congenital infection, leading to persistent infection of numerous organ systems including the central nervous system (CNS). Adult mice persistently infected with LCMV (LCMV-cgPi mice) exhibit learning deficits, such as poor performance in spatial discrimination tests. Given that deficits in spatial learning have been linked to defects in adult neurogenesis, we investigated the impact of congenital LCMV infection on generation of neuroblasts from neural progenitor cells within neurogenic zones of adult mice. In LCMV-cgPi mice, QPCR and immunohistochemistry detected presence of LCMV glycoprotein-coding RNA and nucleoprotein in the hippocampal dentate gyrus and subventricular zone (SVZ), sites of neurogenesis that harbor populations of neuroblasts. Numbers of neuroblasts were reduced in LCMV-cgPi mice, as determined by IHC quantification, and analysis of BrdU incorporation by flow cytometry revealed lower numbers of BrdU-labeled neuroblasts. Additionally, TUNEL assays performed in situ showed increased numbers of apoptotic cells in the two neurogenic regions. Next, neurosphere cultures were infected in vitro with LCMV and differentiated to create a population of cells that consisted of both transit amplifying cells and neuroblasts. Immunocytochemical and TUNEL assays revealed increased numbers of TUNEL-positive cells that express nestin, suggesting that the drop in numbers of neuroblasts was due to a combination of impaired proliferation and apoptosis of progenitor cells. LCMV-cgPi mice exhibited transcriptional up-regulation several cytokines and chemokines, including gamma-interferon inducible chemokines CXCL9 and CXCL10. Chronic up-regulation of these chemokines can facilitate a pro-inflammatory niche that may contribute to defects in neurogenesis.

Organization of multisynaptic inputs to the dorsal and ventral dentate gyrus: retrograde trans-synaptic tracing with rabies virus vector in the rat.

Behavioral, anatomical, and gene expression studies have shown functional dissociations between the dorsal and ventral hippocampus with regard to their involvement in spatial cognition, emotion, and stress. In this study we examined the difference of the multisynaptic inputs to the dorsal and ventral dentate gyrus (DG) in the rat by using retrograde trans-synaptic tracing of recombinant rabies virus vectors. Three days after the vectors were injected into the dorsal or ventral DG, monosynaptic neuronal labeling was present in the entorhinal cortex, medial septum, diagonal band, and supramammillary nucleus, each of which is known to project to the DG directly. As in previous tracing studies, topographical patterns related to the dorsal and ventral DG were seen in these regions. Five days after infection, more of the neurons in these regions were labeled and labeled neurons were also seen in cortical and subcortical regions, including the piriform and medial prefrontal cortices, the endopiriform nucleus, the claustrum, the cortical amygdala, the medial raphe nucleus, the medial habenular nucleus, the interpeduncular nucleus, and the lateral septum. As in the monosynaptically labeled regions, a topographical distribution of labeled neurons was evident in most of these disynaptically labeled regions. These data indicate that the cortical and subcortical inputs to the dorsal and ventral DG are conveyed through parallel disynaptic pathways. This second-order input difference in the dorsal and ventral DG is likely to contribute to the functional differentiation of the hippocampus along the dorsoventral axis.

Borna disease virus-induced neuronal degeneration dependent on host genetic background and prevented by soluble factors.

Infection of newborn rats with Borne disease virus (BDV) results in selective degeneration of granule cell neurons of the dentate gyrus (DG). To study cellular countermechanisms that might prevent this pathology, we screened for rat strains resistant to this BDV-induced neuronal degeneration. To this end, we infected hippocampal slice cultures of different rat strains with BDV and analyzed for the preservation of the DG. Whereas infected cultures of five rat strains, including Lewis (LEW) rats, exhibited a disrupted DG cytoarchitecture, slices of three other rat strains, including Sprague-Dawley (SD), were unaffected. However, efficiency of viral replication was comparable in susceptible and resistant cultures. Moreover, these rat strain-dependent differences in vulnerability were replicated in vivo in neonatally infected LEW and SD rats. Intriguingly, conditioned media from uninfected cultures of both LEW and SD rats could prevent BDV-induced DG damage in infected LEW hippocampal cultures, whereas infection with BDV suppressed the availability of these factors from LEW but not in SD hippocampal cultures. To gain further insights into the genetic basis for this rat strain-dependent susceptibility, we analyzed DG granule cell survival in BDV-infected cultures of hippocampal neurons derived from the F1 and F2 offspring of the crossing of SD and LEW rats. Genome-wide association analysis revealed one resistance locus on chromosome (chr) 6q16 in SD rats and, surprisingly, a locus on chr3q21-23 that was associated with susceptibility. Thus, BDV-induced neuronal degeneration is dependent on the host genetic background and is prevented by soluble protective factors in the disease-resistant SD rat strain.

Assessment of the effects of virus-mediated limited Oct4 overexpression on the structure of the hippocampus and behavior in mice.

Recently, pluripotency induction or cellular reprogramming by introducing critical transcription factors has been extensively studied, but has been demonstrated only in vitro. Based on reports that Oct4 is critically involved in transforming neural stem cells into pluripotent cells, we used the lentiviral vector to introduce the Oct4 gene into the hippocampal dentate gyrus (DG) of adult mice. We examined whether this manipulation led to cellular or behavioral changes, possibly through processes involving the transformation of NS cells into pluripotent cells. The Oct4 lentivirus-infused group and the green fluorescent protein lentivirus-infused group showed a similar thickness of the DG and a comparable level of synaptophysin expression in the DG. Furthermore, our behavioral analyses did not show any differences between the groups concerning exploratory activity, anxiety, or memory abilities. This first trial for pluripotency induction in vivo, despite negative results, provides implications and information for future studies on in vivo cellular reprogramming.

Viral-mediated expression of a constitutively active form of CREB in the dentate gyrus does not induce abnormally enduring fear memory.

Increasing CREB-dependent transcription in dentate gyrus (DG) granule cells in vivo using viral-mediated expression of a constitutively active form of CREB (CREBCA) is sufficient to enhance contextual fear memory but whether this treatment renders memory abnormally enduring is unknown. Here we confirm that over-expressing CREBCA in the DG increases retention of contextual fear conditioning (CFC) and show that this memory decays normally. Specifically, the retention scores of CREBCA mice are significantly higher than those of GFP-infected controls 24h after the conditioning, but match them after a longer exposure session and are still in the same range 48 h later. Our findings provide evidence that boosting selectively CREB activity in the DG promotes the formation of a stronger memory trace but does not increase its resistance to extinguish.

Effects of congenital HCMV infection on synaptic plasticity in dentate gyrus (DG) of rat hippocampus.

This study was carried out to investigate whether the congenital HCMV infection affect the induction and maintenance of LTP /DP. Rat models of Sprague-Dawley rats congenitally infected by HCMV were made. Field excitatory postsynaptic potentials (EPSPs) were recorded in the hippocampal slices of offspring rats (50-65days) to study alterations of LTP /DP in area dentate gyrus (DG) of the hippocampus after congenital infection. The Ca(2+) and mRNA level of calmodulin (CaM) in the hippocampus neurons of the experiment group (congenital infected by HCMV) and the control group were measured;The input/output (I/O) curves of the EPSP slope PS amplitude in area DG in experiment group were significantly depressed when compared to control group (P<0.05). LTP of the EPSP slope and PS amplitude in area DG of the hippocampus was 137±4% (EPSP) and 225±11% (PS) in control rats and 115±9% (EPSP) and 163±7% (PS) in experiment rats (EPSP: F=25.29,P<0.05;PS: F=74.33 P<0.05, two-way ANOVA with Tukey test); DP of the EPSP slope and PS amplitude was 86±3% (EPSP) and 85±2% (PS) in control rats and 94±5% (EPSP) and 93±4% (PS) in congenitally infected rats (EPSP: F=5.62, P<0.05;PS: F=4.22, P<0.05, two-way ANOVA with Tukey test) . At the same time, intracellular [Ca(2+)] and mRNA level of CaM in the hippocampus neurons of the experiment group were significantly increased than that of in the controls ([Ca(2+)]: P<0.01;CaM mRNA: P<0.01) . The results demonstrate that congenital HCMV infection could reduce the range of synaptic plasticity in the Sprague-Dawley rats, which may trigger the dysfunction of learning and memory through disrupting the calcium balance.

Interleukin-1 mediates long-term hippocampal dentate granule cell loss following postnatal viral infection.

Viral infections of the developing CNS can cause long-term neuropathological sequela through undefined mechanisms. Proinflammatory cytokines such as IL-1beta have gained attention in mediating neurodegeneration in corticohippocampal structures due to a variety of insults in adults, though there is less information on the developing brain. Little is known concerning the spatial-temporal pattern of IL-1beta induction in the developing hippocampus following live virus infection, and there are few studies addressing the long-term consequences of this cytokine induction. We report that infection of rats with lymphocytic choriomeningitis virus on postnatal day 4 induces IL-1beta protein in select regions of the hippocampus on 6, 15, 21, and 45 days after infection. This infection resulted in a 71% reduction of dentate granule cell neurons by the time the rats reached mid-adulthood. We further investigated the causative role of IL-1 in this dentate granule cell loss by blocking IL-1 activity using an IL-1ra-expressing adenoviral vector administered at the time of infection. Blockade of IL-1 abrogated the infection-associated neuron loss in this vivo model. Considering that IL-1 can be triggered by multiple perinatal insults, our findings suggest that early therapy with anti-inflammatory agents that block IL-1 may be effective for reducing adulthood neuropathology.

Primary target cells of herpes simplex virus type 1 in the hippocampus.

Herpes simplex virus type 1 (HSV-1) causes fatal and sporadic encephalitis in human. The encephalitis-survivors frequently suffer from symptoms of memory deficits. It remains unclear how HSV-1 induces tissue damages in memory formation-associated brain tissues such as the hippocampus. In this study, we examined HSV-1 infection in the hippocampus using a rat HSV-1 infection model. We found profound pathological changes in the hippocampus and large numbers of HSV-1 antigen-positive cells in the dentate gyrus (DG) subfield of HSV-1-infected rats. To understand the precise mechanism of HSV-1-induced tissue damages in the hippocampus, we employed rat organotypic hippocampal slice cultures (OHC) as an in vitro HSV-1 infection model. In OHC, HSV-1 infection predominated in neuronal cells and the infected neuronal cells were severely damaged. Longitudinal analysis indicated that granule cells in DG subfield were extremely vulnerable to HSV-1 infection among neuronal cells in the hippocampus. Since DG granule cells play a crucial role in memory formation, disruption of these cells may be a primary step leading to memory deficits.

Neurons born in the adult dentate gyrus form functional synapses with target cells.

Adult neurogenesis occurs in the hippocampus and the olfactory bulb of the mammalian CNS. Recent studies have demonstrated that newborn granule cells of the adult hippocampus are postsynaptic targets of excitatory and inhibitory neurons, but evidence of synapse formation by the axons of these cells is still lacking. By combining retroviral expression of green fluorescent protein in adult-born neurons of the mouse dentate gyrus with immuno-electron microscopy, we found output synapses that were formed by labeled terminals on appropriate target cells in the CA3 area and the hilus. Furthermore, retroviral expression of channelrhodopsin-2 allowed us to light-stimulate newborn granule cells and identify postsynaptic target neurons by whole-cell recordings in acute slices. Our structural and functional evidence indicates that axons of adult-born granule cells establish synapses with hilar interneurons, mossy cells and CA3 pyramidal cells and release glutamate as their main neurotransmitter.

Hippocampal poly(ADP-Ribose) polymerase 1 and caspase 3 activation in neonatal bornavirus infection.

Infection of neonatal rats with Borna disease virus results in a characteristic behavioral syndrome and apoptosis of subsets of neurons in the hippocampus, cerebellum, and cortex (neonatal Borna disease [NBD]). In the NBD rat hippocampus, dentate gyrus granule cells progressively degenerate. Apoptotic loss of granule cells in NBD is associated with accumulation of zinc in degenerating neurons and reduced zinc in granule cell mossy fibers. Excess zinc can trigger poly(ADP-ribose) polymerase 1 (PARP-1) activation, and PARP-1 activation can mediate neuronal death. Here, we evaluate hippocampal PARP-1 mRNA and protein expression levels, activation, and cleavage, as well as apoptosis-inducing factor (AIF) nuclear translocation and executioner caspase 3 activation, in NBD rats. PARP-1 mRNA and protein levels were increased in NBD hippocampi. PARP-1 expression and activity were increased in granule cell neurons and glia with enhanced ribosylation of proteins, including PARP-1 itself. In contrast, levels of poly(ADP-ribose) glycohydrolase mRNA were decreased in NBD hippocampi. PARP-1 cleavage and AIF expression were also increased in astrocytes in NBD hippocampi. Levels of activated caspase 3 protein were increased in NBD hippocampi and localized to nuclei, mossy fibers, and dendrites of granule cell neurons. These results implicate aberrant zinc homeostasis, PARP-1, and caspase 3 activation as contributing factors in hippocampal neurodegeneration in NBD.

Viral risk factor for seizures: pathobiology of dynorphin in herpes simplex viral (HSV-1) seizures in an animal model.

Up to 89% of patients with herpes simplex virus type-1 (HSV-1) encephalitis can have seizures. Possibly, viruses are environmental triggers for seizures in genetically vulnerable individuals. Inherited dynorphin promoter polymorphisms are associated with temporal lobe epilepsy and febrile seizures in man. In animals, the dynorphin system in the hippocampus regulates excitability. The hypothesis that reduced dynorphin expression in dentate gyrus of hippocampus due to HSV-1 infection leads to epileptic responses was tested in a rat model of HSV-1 encephalitis using EEG recording, histopathological and neuropharmacologic probes. HSV-1 infection causes loss of dynorphin A-like immunoreactivity in hippocampus, an effect independent of direct viral interference and cell loss. A kappa opioid receptor agonist U50488 effectively blocks ictal activity, linking absence of dynorphin to propensity for epileptic activity. These findings show a vulnerability of hippocampal dynorphin during infection, suggesting a neurochemical basis for seizures that may be generalizable to other encephalitic viruses.

Neonatal viral infection decreases neuronal progenitors and impairs adult neurogenesis in the hippocampus.

While adult neurogenesis has been demonstrated in the hippocampal dentate gyrus of several mammalian species, including humans, the impact of viral infections has not been well studied. To examine this question we used a model in which neonatal rats are infected with lymphocytic choriomeningitis virus (LCMV) leading to a gradual loss of dentate granule cells (DGCs), which becomes fully evident in adulthood. Stereological cell counts performed 8 months after infection revealed that the loss of mature DGCs was accompanied by an 84.2% reduction in proliferation of DGCs as measured by BrdU uptake. Moreover, there was a severe loss of Mash1-labeled neuronal progenitor cells (87 and 83% decrease in the granule cell layer and hilus, respectively). Thus, neurogenesis is impaired in this model of chronic DGC loss, perhaps due to a virus-induced impoverishment of DGC neuronal progenitors. The LCMV model could be exploited to examine pathophysiological mechanisms of neurodegeneration and to test pharmacological strategies aimed at increasing neurogenesis or rescuing multipotent progenitors.

Viral infection of developing GABAergic neurons in a model of hippocampal disinhibition.

Mechanisms by which perinatal viral infections can disrupt hippocampal development and cause selective neuronal death may have implications for temporal lobe epilepsy and schizophrenia. Despite abnormalities of inhibitory interneurons in these diseases, the causal relationships between such neurotransmitter changes and viral infections remain unclear. This relationship was examined in a model in which rats, infected with lymphocytic choriomeningitis virus (LCMV) as neonates, manifest a gradual loss of hippocampal dentate granule cells and neuronal hyperexcitability. The current data demonstrate that GABAergic interneurons are dual immunostained for LCMV antigens prior to the loss of dentate granule cells, supporting the hypothesis that LCMV may disrupt developing inhibitory circuits causing unbalanced excitatory neurotransmission and the eventual death of dentate granule cells due to excitotoxicity.

Apoptosis in experimental rabies in bax-deficient mice.

The challenge virus standard (CVS) of fixed rabies virus produces a fatal encephalitis in adult and suckling mice after intracerebral inoculation. The infection is associated with apoptotic cell death in brain neurons and increased immunoreactivity to the Bax protein in the hippocampus and cerebral cortex. Five- to 7-day-old bax-deficient mice and their wild-type littermates were inoculated intracerebrally with either CVS or the RV194-2 variant of rabies virus, which is avirulent in adult mice after intracerebral inoculation. The clinical disease was similar with both viruses in bax-deficient and wild-type mice with 100% mortality. CVS produced similar apoptotic changes in bax-deficient and wild-type mice, except that apoptosis was more marked in neurons of the dentate gyrus and cortical neurons in the wild-type mice. After inoculation with RV194-2, the morphologic changes of apoptosis were markedly less severe in the cerebral cortex, hippocampus, and cerebellum of the bax-deficient mice than wild-type mice. However, apoptotic changes were moderate to severe in the brain stem in both wild-type and bax-deficient mice with both viruses. Although apoptotic cell death was much less prominent in bax-deficient mice after inoculation with RV194-2, apoptosis of infected brain stem neurons occurred in this fatal infection. Although the Bax protein plays an important role in modulating rabies virus-induced apoptosis under specific experimental conditions, other modulators are also likely important.

Adenovirus-mediated gene transfer in neurons: construction and characterization of a vector for heterologous expression of the axonal cell adhesion molecule axonin-1.

By homologous recombination, a first-generation adenovirus-based gene transfer vector, AdCMVax-1, was constructed as a means of manipulating the expression level of the axonal cell adhesion molecule axonin-1 in neurons and glial cells. AdCMVax-1 harbours the entire coding region of the chicken axonin-1 cDNA under the transcriptional control of the Cytomegalovirus enhancer/promoter in the early-region 1 of the viral genome. Characterization of AdCMVax-1 in vitro revealed highly efficient gene transfer and expression of recombinant axonin-1 in neurons and glial cells of dissociated rat dorsal root ganglia. Similar to its native counterpart, virus-derived axonin-1 was detected on the cell body, neurites, and growth cones of transduced neurons, occurred in a secreted and membrane-associated form, and could be cleaved from the membrane with phosphatidylinositol-specific phospholipase C. Functional characterization of recombinant axonin-1 revealed the same binding properties as previously reported for native axonin-1 isolated from the vitreous fluid of chicken embryos. In vivo gene transfer was studied by stereotactic injection of AdCMVax-1 in the dentate gyrus of the hippocampus and the facial nucleus in the brainstem of adult Wistar rats and revealed high level expression of recombinant axonin-1 in a subset of hippocampal neurons and motor neurons in the facial nucleus.