Replication of early and recent Zika virus isolates throughout mouse brain development.

Fetal infection with Zika virus (ZIKV) can lead to congenital Zika virus syndrome (cZVS), which includes cortical malformations and microcephaly. The aspects of cortical development that are affected during virus infection are unknown. Using organotypic brain slice cultures generated from embryonic mice of various ages, sites of ZIKV replication including the neocortical proliferative zone and radial columns, as well as the developing midbrain, were identified. The infected radial units are surrounded by uninfected cells undergoing apoptosis, suggesting that programmed cell death may limit viral dissemination in the brain and may constrain virus-associated injury. Therefore, a critical aspect of ZIKV-induced neuropathology may be defined by death of uninfected cells. All ZIKV isolates assayed replicated efficiently in early and midgestation cultures, and two isolates examined replicated in late-gestation tissue. Alteration of neocortical cytoarchitecture, such as disruption of the highly elongated basal processes of the radial glial progenitor cells and impairment of postmitotic neuronal migration, were also observed. These data suggest that all lineages of ZIKV tested are neurotropic, and that ZIKV infection interferes with multiple aspects of neurodevelopment that contribute to the complexity of cZVS.

Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex.

The recent Zika outbreak in South America and French Polynesia was associated with an epidemic of microcephaly, a disease characterized by a reduced size of the cerebral cortex. Other members of the Flavivirus genus, including West Nile virus (WNV), can cause encephalitis but were not demonstrated to cause microcephaly. It remains unclear whether Zika virus (ZIKV) and other flaviviruses may infect different cell populations in the developing neocortex and lead to distinct developmental defects. Here, we describe an assay to infect mouse E15 embryonic brain slices with ZIKV, WNV and dengue virus serotype 4 (DENV-4). We show that this tissue is able to support viral replication of ZIKV and WNV, but not DENV-4. Cell fate analysis reveals a remarkable tropism of ZIKV infection for neural stem cells. Closely related WNV displays a very different tropism of infection, with a bias towards neurons. We further show that ZIKV infection, but not WNV infection, impairs cell cycle progression of neural stem cells. Both viruses inhibited apoptosis at early stages of infection. This work establishes a powerful comparative approach to identify ZIKV-specific alterations in the developing neocortex and reveals specific preferential infection of neural stem cells by ZIKV.

Targeting single neuronal networks for gene expression and cell labeling in vivo.

To understand fine-scale structure and function of single mammalian neuronal networks, we developed and validated a strategy to genetically target and trace monosynaptic inputs to a single neuron in vitro and in vivo. The strategy independently targets a neuron and its presynaptic network for specific gene expression and fine-scale labeling, using single-cell electroporation of DNA to target infection and monosynaptic retrograde spread of a genetically modifiable rabies virus. The technique is highly reliable, with transsynaptic labeling occurring in every electroporated neuron infected by the virus. Targeting single neocortical neuronal networks in vivo, we found clusters of both spiny and aspiny neurons surrounding the electroporated neuron in each case, in addition to intricately labeled distal cortical and subcortical inputs. This technique, broadly applicable for probing and manipulating single neuronal networks with single-cell resolution in vivo, may help shed new light on fundamental mechanisms underlying circuit development and information processing by neuronal networks throughout the brain.

Antisense in vivo knockdown of synaptotagmin I by HVJ-liposome mediated gene transfer attenuates ischemic brain damage in neonatal rats.

Synaptic release of the excitatory amino acid glutamate is considered as an important mechanism in the pathogenesis of ischemic brain damage in neonates. Synaptotagmin I is one of exocytosis-related proteins at nerve terminals and considered to accelerate the exocytosis of synaptic vesicles by promoting fusion between the vesicles and plasma membrane. To test the possibility that antisense in vivo knockdown of synaptotagmin I modulates the exocytotic release of glutamate, thus suppressing the excitotoxic intracellular processes leading to neuronal death following ischemia in the neonatal brain, we injected antisense oligodeoxynucleotides (ODNs) targeting synaptotagmin I (0.3 (AS), 0.15 (0.5 AS), or 0.03 microg (0.1 AS), or vehicle) into the lateral ventricles of 7-day-old rats by using a hemagglutinating virus of Japan (HVJ)-liposome mediated gene transfer technique. At 10 days of age, these rats were subjected to an electrical coagulation of the right external and internal carotid arteries, then the insertion of a solid nylon thread into the right common carotid artery toward the ascending aorta up to 10-12 mm from the upper edge of the sternocleidomastoid muscle. Cerebral ischemia was induced by clamping the left external and internal carotid arteries with a clip, and ended by removing the clip 2h later. Twenty-four hours after the end of ischemia, the extent of ischemic brain damage was neuropathologically and quantitatively evaluated in the neocortex and striatum. While the relative volume of damage in the cerebral cortex and striatum of the vehicle group was extended to 40% and 13.7%, respectively, that in the AS group was significantly reduced to 4.8% and 0.6%. In the 0.5 AS group, the relative volume of ischemic damage in the cerebral cortex and striatum was reduced to 20.5% and 15.4%, respectively, and the difference between the 0.5 AS group and vehicle group was statistically significant in the neocortex, but not in the striatum. These results indicated that antisense in vivo knockdown of synaptotagmin I successfully attenuated ischemic brain damage in neonatal rats and that the effect was dose-dependent. It was also suggested that this treatment was more effective in the neocortex than in the striatum in neonatal rats.

Viral regulation of aquaporin 4, connexin 43, microcephalin and nucleolin.

The current study investigated whether human influenza viral infection in midpregnancy leads to alterations in proteins involved in brain development. Human influenza viral infection was administered to E9 pregnant Balb/c mice. Brains of control and virally-exposed littermates were subjected to microarray analysis, SDS-PAGE and western blotting at three postnatal stages. Microarray analysis of virally-exposed mouse brains showed significant, two-fold change in expression of multiple genes in both neocortex and cerebellum when compared to sham-infected controls. Levels of mRNA and protein levels of four selected genes were examined in brains of exposed mice. Nucleolin mRNA was significantly decreased in day 0 and day 35 neocortex and significantly increased in day 35 cerebellum. Protein levels were significantly upregulated at days 35 and 56 in neocortex and at day 56 in cerebellum. Connexin 43 protein levels were significantly decreased at day 56 in neocortex. Aquaporin 4 mRNA was significantly decreased in day 0 neocortex. Aquaporin 4 protein levels decreased in neocortex significantly at day 35. Finally, microcephalin mRNA was significantly decreased in day 56 neocortex and protein levels were significantly decreased at 56 cerebellum. These data suggest that influenza viral infection in midpregnancy in mice leads to long-term changes in brain markers for enhanced ribosome genesis (nucleolin), increased production of immature neurons (microcephalin), and abnormal glial-neuronal communication and neuron migration (connexin 43 and aquaporin 4).

Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter.

Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an approximately 9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported approximately 90% glutamatergic neuron-specific expression. The GAD67 promoter supported approximately 90% GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine a glutamatergic or GABAergic neuron.

Fusion of microglia with pyramidal neurons after retroviral infection.

The neurogenic potential of the postnatal neocortex has not been tested previously with a combination of both retroviral and bromodeoxyuridine (BrdU) labeling. Here we report that injections of enhanced green fluorescent protein (eGFP) retrovirus into 134 postnatal rats resulted in GFP labeling of 642 pyramidal neurons in neocortex. GFP-labeled neocortical pyramidal neurons, however, unlike GFP-labeled glia, did not incorporate BrdU. Closer inspection of retrovirally labeled neurons revealed microglia fused to the apical dendrites of labeled pyramidal neurons. Retroviral infection of mixed cultures of cortical neurons and glia confirmed the presence of specific neuronal-microglial fusions. Microglia did not fuse to other glial cell types, and cultures not treated with retrovirus lacked microglial-neuronal fusion. Furthermore, activation of microglia by lipopolysaccharide greatly increased the virally induced fusion of microglia to neurons in culture. These results indicate a novel form of specific cell fusion between neuronal dendrites and microglia and further illustrate the need for caution when interpreting evidence for neuronogenesis in the postnatal brain.

Plastic reorganization of hippocampal and neocortical circuitry in experimental traumatic brain injury in the immature rat.

The reorganization of circuitry in the immature forebrain resulting from controlled cortical impact was examined with viral transneuronal tracing. Animals injured on postnatal day (PND) 17 and sham controls from the same litters received an intracerebral injection of a recombinant strain of pseudorabies virus (PRV) into the entorhinal cortex on PND 45. Fifty hours following injection of virus the animals were perfused and infected neurons were localized immunohistochemically with antisera specific for PRV. Prior studies have demonstrated that the PRV recombinant used in this analysis moves exclusively in the retrograde direction through synaptically linked neurons. CCI induced a necrotic loss of cortex at the site of impact and variable damage to the underlying corpus callosum and rostral (dorsal) hippocampus that was not present in sham controls. Analysis of viral transport in sham controls revealed retrograde transport of virus through hippocampal and neocortical circuitry in a pattern consistent with established patterns of connectivity and topography. Injured animals exhibited preservation of topographically organized connections in both the hippocampus and neocortex. However, the magnitude of labeling in the injured hemisphere was significantly increased relative to control animals and correlated with the magnitude of the injury. The distribution of infected neurons in the contralateral uninjured hemisphere also conformed to known connections. However differences in the involvement of the corpus callosum in the injury resulted in greater variability in the number of infected neurons among cases. These data provide novel insights into trauma induced reorganization of the developing brain and add to the experimental tools that can be used to assess the basis for functional recovery in animal models of developmental traumatic brain injury.

Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors.

BACKGROUND: Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain. RESULTS: Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. CONCLUSION: AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.

Upregulation of COX-2 and CGRP expression in resident cells of the Borna disease virus-infected brain is dependent upon inflammation.

Infection of immunocompetent adult rats with Borna disease virus (BDV) causes severe encephalitis and neural dysfunction. The expression of COX-2 and CGRP, genes previously shown to be implicated in CNS disease and peripheral inflammation, was dramatically upregulated in the cortical neurons of acutely BDV-infected rats. Neuronal COX-2 and CGRP upregulation was predominantly seen in brain areas where ED1-positive macrophages/microglia accumulated. In addition, COX-2 expression was strongly induced in brain endothelial cells and the number of COX-2 immunoreactive microglial cells was increased. In contrast, despite increased expression of viral antigens, neither COX-2 nor CGRP expression was altered in the CNS of BDV-infected rats treated with dexamethasone, or tolerant to BDV. Thus, increased CGRP and COX-2 expression in the BDV-infected brain is the result of the inflammatory response and likely to be involved in the pathogenesis of virus-induced encephalitis.

HIV encephalitis, proviral load and dementia in drug users and homosexuals with AIDS. Effect of neocortical involvement.

In this consecutive autopsy study, the pathological evidence of HIV encephalitis, which included the presence of giant cells and/or HIV p24 immunopositivity, was found more frequently in drug users (25 of 45; 56%) than in homosexual men (6 of 35; 17%) with AIDS (P < 0.01). Productive infection, as shown by HIV p24 positivity, was found in frontal lobe white matter in 29 of the 31 HIV encephalitis cases, but was also present in grey matter in 50% of the HIV encephalitis cases. Immunopositivity was confined to microglia, monocytes and most but not all giant cells. HIV-1 proviral load was determined by quantitative PCR in 65 of the 80 cases (separately in grey and white matter in 49 of these), and correlated well with the presence of HIV encephalitis (P < 0.001). Twenty-five patients with AIDS (13 male homosexuals, 12 drug users) showed no HIV encephalitis, opportunistic infection or cerebral lymphoma, while 18 (2 male homosexuals, 16 drug users) showed pure HIV encephalitis. Cognitive function had been assessed prospectively in this cohort and graded as normal or mildly, moderately or severely impaired. Because opportunistic infections and lymphomas of the brain may also lead to dementia, patients found to have these conditions at autopsy were excluded from the final analysis of the cases with dementia, so that the precise correlation between cognitive impairment and pure HIV encephalitis could be determined in this cohort without possible confounding variables. Fourteen of 18 patients with pure HIV encephalitis had shown cognitive impairment. Severe dementia correlated better with pure HIV encephalitis in cases in which grey matter involvement was present (7 out of 9) than in those in which only white matter was involved (2 out of 9) (P < 0.05), although milder degrees of cognitive impairment had been present in a further 5 HIV encephalitis cases. No correlation was found between zidovudine therapy and the degree of cognitive impairment. Systemic and cerebral opportunistic infections and lymphoma showed a negative association with HIV encephalitis, being more common in homosexuals than in drug users, despite comparable CD4 counts in the two groups. These findings suggest that neocortical productive HIV infection is a significant factor in AIDS-related dementia, although this may reflect merely a higher overall viral burden in the brain.