Brain exposure to SARS-CoV-2 virions perturbs synaptic homeostasis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with short- and long-term neurological complications. The variety of symptoms makes it difficult to unravel molecular mechanisms underlying neurological sequalae after coronavirus disease 2019 (COVID-19). Here we show that SARS-CoV-2 triggers the up-regulation of synaptic components and perturbs local electrical field potential. Using cerebral organoids, organotypic culture of human brain explants from individuals without COVID-19 and post-mortem brain samples from individuals with COVID-19, we find that neural cells are permissive to SARS-CoV-2 to a low extent. SARS-CoV-2 induces aberrant presynaptic morphology and increases expression of the synaptic components Bassoon, latrophilin-3 (LPHN3) and fibronectin leucine-rich transmembrane protein-3 (FLRT3). Furthermore, we find that LPHN3-agonist treatment with Stachel partially restored organoid electrical activity and reverted SARS-CoV-2-induced aberrant presynaptic morphology. Finally, we observe accumulation of relatively static virions at LPHN3-FLRT3 synapses, suggesting that local hindrance can contribute to synaptic perturbations. Together, our study provides molecular insights into SARS-CoV-2-brain interactions, which may contribute to COVID-19-related neurological disorders.

Neurodegenerative phagocytes mediate synaptic stripping in Neuro-HIV.

Glial cell activation is a hallmark of several neurodegenerative and neuroinflammatory diseases. During HIV infection, neuroinflammation is associated with cognitive impairment, even during sustained long-term suppressive antiretroviral therapy. However, the cellular subsets contributing to neuronal damage in the CNS during HIV infection remain unclear. Using post-mortem brain samples from eight HIV patients and eight non-neurological disease controls, we identify a subset of CNS phagocytes highly enriched in LGALS3, CTSB, GPNMB and HLA-DR, a signature identified in the context of ageing and neurodegeneration. In HIV patients, the presence of this phagocyte phenotype was associated with synaptic stripping, suggesting an involvement in the pathogenesis of HIV-associated neurocognitive disorder. Taken together, our findings elucidate some of the molecular signatures adopted by CNS phagocytes in HIV-positive patients and contribute to the understanding of how HIV might pave the way to other forms of cognitive decline in ageing HIV patient populations.

Chronic SSRI treatment reverses HIV-1 protein-mediated synaptodendritic damage.

HIV-1 infection affects approximately 37 million individuals, and approximately 50% of seropositive individuals will develop symptoms of clinical depression and/or apathy. Dysfunctions of both serotonergic and dopaminergic neurotransmission have been implicated in the pathogenesis of motivational alterations. The present study evaluated the efficacy of a SSRI (escitalopram) in the HIV-1 transgenic (Tg) rat. Behavioral, neurochemical, and neuroanatomical outcomes with respect to HIV-1 and sex were evaluated to determine the efficacy of chronic escitalopram treatment. Escitalopram treatment restored function in each of the behavioral tasks that were sensitive to HIV-1-induced impairments. Further, escitalopram treatment restored HIV-1-mediated synaptodendritic damage in the nucleus accumbens; treatment with escitalopram significantly increased dendritic proliferation in HIV-1 Tg rats. However, restoration did not consistently occur with the neurochemical analysis in the HIV-1 rat. Taken together, these results suggest a role for SSRI therapies in repairing long-term HIV-1 protein-mediated neuronal damage and restoring function.

Evidence of Coronavirus (CoV) Pathogenesis and Emerging Pathogen SARS-CoV-2 in the Nervous System: A Review on Neurological Impairments and Manifestations.

The coronavirus disease 2019 (COVID-19) pandemic is an issue of global significance that has taken the lives of many across the world. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for its pathogenesis. The pulmonary manifestations of COVID-19 have been well described in the literature. Initially, it was thought to be limited to the respiratory system; however, we now recognize that COVID-19 also affects several other organs, including the nervous system. Two similar human coronaviruses (CoV) that cause severe acute respiratory syndrome (SARS-CoV-1) and Middle East respiratory syndrome (MERS-CoV) are also known to cause disease in the nervous system. The neurological manifestations of SARS-CoV-2 infection are growing rapidly, as evidenced by several reports. There are several mechanisms responsible for such manifestations in the nervous system. For instance, post-infectious immune-mediated processes, direct virus infection of the central nervous system (CNS), and virus-induced hyperinflammatory and hypercoagulable states are commonly involved. Guillain-Barré syndrome (GBS) and its variants, dysfunction of taste and smell, and muscle injury are numerous examples of COVID-19 PNS (peripheral nervous system) disease. Likewise, hemorrhagic and ischemic stroke, encephalitis, meningitis, encephalopathy acute disseminated encephalomyelitis, endothelialitis, and venous sinus thrombosis are some instances of COVID-19 CNS disease. Due to multifactorial and complicated pathogenic mechanisms, COVID-19 poses a large-scale threat to the whole nervous system. A complete understanding of SARS-CoV-2 neurological impairments is still lacking, but our knowledge base is rapidly expanding. Therefore, we anticipate that this comprehensive review will provide valuable insights and facilitate the work of neuroscientists in unfolding different neurological dimensions of COVID-19 and other CoV associated abnormalities.

Zika virus replicates in adult human brain tissue and impairs synapses and memory in mice.

Neurological complications affecting the central nervous system have been reported in adult patients infected by Zika virus (ZIKV) but the underlying mechanisms remain unknown. Here, we report that ZIKV replicates in human and mouse adult brain tissue, targeting mature neurons. ZIKV preferentially targets memory-related brain regions, inhibits hippocampal long-term potentiation and induces memory impairment in adult mice. TNF-α upregulation, microgliosis and upregulation of complement system proteins, C1q and C3, are induced by ZIKV infection. Microglia are found to engulf hippocampal presynaptic terminals during acute infection. Neutralization of TNF-α signaling, blockage of microglial activation or of C1q/C3 prevent synapse and memory impairment in ZIKV-infected mice. Results suggest that ZIKV induces synapse and memory dysfunction via aberrant activation of TNF-α, microglia and complement. Our findings establish a mechanism by which ZIKV affects the adult brain, and point to the need of evaluating cognitive deficits as a potential comorbidity in ZIKV-infected adults.

SIV-Mediated Synaptic Dysfunction Is Associated with an Increase in Synapsin Site 1 Phosphorylation and Impaired PP2A Activity.

Although the reduction of viral loads in people with HIV undergoing combination antiretroviral therapy has mitigated AIDS-related symptoms, the prevalence of neurological impairments has remained unchanged. HIV-associated CNS dysfunction includes impairments in memory, attention, memory processing, and retrieval. Here, we show a significant site-specific increase in the phosphorylation of Syn I serine 9, site 1, in the frontal cortex lysates and synaptosome preparations of male rhesus macaques infected with simian immunodeficiency virus (SIV) but not in uninfected or SIV-infected antiretroviral therapy animals. Furthermore, we found that a lower protein phosphatase 2A (PP2A) activity, a phosphatase responsible for Syn I (S9) dephosphorylation, is primarily associated with the higher S9 phosphorylation in the frontal cortex of SIV-infected macaques. Comparison of brain sections confirmed higher Syn I (S9) in the frontal cortex and greater coexpression of Syn I and PP2A A subunit, which was observed as perinuclear aggregates in the somata of the frontal cortex of SIV-infected macaques. Synaptosomes from SIV-infected animals were physiologically tested using a synaptic vesicle endocytosis assay and FM4-64 dye showing a significantly higher baseline depolarization levels in synaptosomes of SIV+-infected than uninfected control or antiretroviral therapy animals. A PP2A-activating FDA-approved drug, FTY720, decreased the higher synaptosome depolarization in SIV-infected animals. Our results suggest that an impaired distribution and lower activity of serine/threonine phosphatases in the context of HIV infection may cause an indirect effect on the phosphorylation levels of essential proteins involving in synaptic transmission, supporting the occurrence of specific impairments in the synaptic activity during SIV infection.SIGNIFICANCE STATEMENT Even with antiretroviral therapy, neurocognitive deficits, including impairments in attention, memory processing, and retrieval, are still major concerns in people living with HIV. Here, we used the rhesus macaque simian immunodeficiency virus model with and without antiretroviral therapy to study the dynamics of phosphorylation of key amino acid residues of synapsin I, which critically impacts synaptic vesicle function. We found a significant increase in synapsin I phosphorylation at serine 9, which was driven by dysfunction of serine/threonine protein phosphatase 2A in the nerve terminals. Our results suggest that an impaired distribution and lower activity of serine/threonine phosphatases in the context of HIV infection may cause an indirect effect on the phosphorylation levels of essential proteins involved in synaptic transmission.

Hitchhiking on the neuronal highway: Mechanisms of transsynaptic specificity.

Transsynaptic viral tracers are an invaluable neuroanatomical tool to define neuronal circuit connectivity across single or multiple synapses. There are variants that label either inputs or outputs of defined starter populations, most of which are based on the herpes and rabies viruses. However, we still have an incomplete understanding of the factors influencing specificity of neuron-neuron transmission and labeling of inputs vs. outputs. This article will touch on three topics: First, how specific are the directional transmission patterns of these viruses? Second, what are the properties that confer synaptic specificity of viral transmission? Lastly, what can we learn from this specificity, and can we use it to devise better transsynaptic tracers?

Scaling Synapses in the Presence of HIV.

A defining feature of HIV-associated neurocognitive disorder (HAND) is the loss of excitatory synaptic connections. Synaptic changes that occur during exposure to HIV appear to result, in part, from a homeostatic scaling response. Here we discuss the mechanisms of these changes from the perspective that they might be part of a coping mechanism that reduces synapses to prevent excitotoxicity. In transgenic animals expressing the HIV proteins Tat or gp120, the loss of synaptic markers precedes changes in neuronal number. In vitro studies have shown that HIV-induced synapse loss and cell death are mediated by distinct mechanisms. Both in vitro and animal studies suggest that HIV-induced synaptic scaling engages new mechanisms that suppress network connectivity and that these processes might be amenable to therapeutic intervention. Indeed, pharmacological reversal of synapse loss induced by HIV Tat restores cognitive function. In summary, studies indicate that there are temporal, mechanistic and pharmacological features of HIV-induced synapse loss that are consistent with homeostatic plasticity. The increasingly well delineated signaling mechanisms that regulate synaptic scaling may reveal pharmacological targets suitable for normalizing synaptic function in chronic neuroinflammatory states such as HAND.

HIV gp120-induced neuroinflammation potentiates NMDA receptors to overcome basal suppression of inhibitory synapses by p38 MAPK.

HIV-associated neurocognitive disorder affects about half of HIV-infected patients. HIV impairs neuronal function through indirect mechanisms mainly mediated by inflammatory cytokines and neurotoxic viral proteins, such as the envelope protein gp120. HIV gp120 elicits a neuroinflammatory response that potentiates NMDA receptor function and induces the loss of excitatory synapses. How gp120 influences neuronal inhibition remains unknown. In this study, we expressed a green fluorescent protein (GFP)-tagged recombinant antibody-like protein that binds to the post-synaptic scaffolding protein gephyrin to label inhibitory synapses in living neurons. Treatment with 600 pM gp120 for 24 h increased the number of labeled inhibitory synapses. HIV gp120 evoked the release of interleukin-1ß (IL-1ß) from microglia to activate IL-1 receptors on neurons. Subsequent activation of the tyrosine kinase Src and GluN2A-containing NMDA receptors increased the number of inhibitory synapses via a process that required protein synthesis. In naïve cultures, inhibition of neuronal p38 mitogen-activated protein kinase (p38 MAPK) increased the number of inhibitory synapses suggesting that p38 MAPK produces a basal suppression of inhibitory synapses that is overcome in the presence of gp120. Direct activation of a mutant form of p38 MAPK expressed in neurons mimicked basal suppression of inhibitory synapses. This study shows for the first time that gp120-induced neuroinflammation increases the number of inhibitory synapses and that this increase overcomes a basal suppression of synaptic inhibition. Increased inhibition may be an adaptive mechanism enabling neurons to counteract excess excitatory input in order to maintain network homeostasis. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

HIV-1 cell-to-cell transmission and broadly neutralizing antibodies.

HIV-1 spreads through contacts between infected and target cells. Polarized viral budding at the contact site forms the virological synapse. Additional cellular processes, such as nanotubes, filopodia, virus accumulation in endocytic or phagocytic compartments promote efficient viral propagation. Cell-to-cell transmission allows immune evasion and likely contributes to HIV-1 spread in vivo. Anti-HIV-1 broadly neutralizing antibodies (bNAbs) defeat the majority of circulating viral strains by binding to the viral envelope glycoprotein (Env). Several bNAbs have entered clinical evaluation during the last years. It is thus important to understand their mechanism of action and to determine how they interact with infected cells. In experimental models, HIV-1 cell-to-cell transmission is sensitive to neutralization, but the effect of antibodies is often less marked than during cell-free infection. This may be due to differences in the conformation or accessibility of Env at the surface of virions and cells. In this review, we summarize the current knowledge on HIV-1 cell-to-cell transmission and discuss the role of bNAbs during this process.

Simulations of centriole of polarized centrosome as a monopole antenna in immune and viral synapses.

The immune synapse (IS) is a temporary interface between an antigen-presenting cell and an effector lymphocyte. Viral synapse is a molecularly organized cellular junction that is structurally similar to the IS. Primary cilium is considered as a functional homologue of the IS due to the morphological and functional similarities in architecture between both micotubule structures. It has been hypothesized that endogenous electromagnetic field in the cell is generated by a unique cooperating system between mitochondria and microtubules. We are extending this prior hypothesis of the endogenous electromagnetic field in the cell postulating that polarized centriole in immune and viral synapse could serve as a monopole antenna. This is an addition to our hypothesis that primary cilium could serve as a monopole antenna. We simulated the distribution of electric field of centriole of polarized centrosome as a monopole antenna in immune and viral synapse. Very weak electromagnetic field of polarized centriole of CD8+ T lymphocyte in IS can contribute to the transport of cytolytic granules into the attacked (cancer) cell. Analogically, very weak electromagnetic field of polarized centriole in viral synapse of infected CD4 cells can aid the transport of viruses (human immunodeficiency virus) to non-infected CD4 cells. We hypothesized that healthy organisms need these monopole antennas. If, during the neoplastic transformation, healthy cells lose monopole antennas in form of primary cilia, the IS aims to replace them by monopole antennas of polarized centrioles in IS to restore homeostasis.

Neurotropism and behavioral changes associated with Zika infection in the vector Aedes aegypti.

Understanding Zika virus infection dynamics is essential, as its recent emergence revealed possible devastating neuropathologies in humans, thus causing a major threat to public health worldwide. Recent research allowed breakthrough in our understanding of the virus and host pathogenesis; however, little is known on its impact on its main vector, Aedes aegypti. Here we show how Zika virus targets Aedes aegypti's neurons and induces changes in its behavior. Results are compared to dengue virus, another flavivirus, which triggers a different pattern of behavioral changes. We used microelectrode array technology to record electrical spiking activity of mosquito primary neurons post infections and discovered that only Zika virus causes an increase in spiking activity of the neuronal network. Confocal microscopy also revealed an increase in synapse connections for Zika virus-infected neuronal networks. Interestingly, the results also showed that mosquito responds to infection by overexpressing glutamate regulatory genes while maintaining virus levels. This neuro-excitation, possibly via glutamate, could contribute to the observed behavioral changes in Zika virus-infected Aedes aegypti females. This study reveals the importance of virus-vector interaction in arbovirus neurotropism, in humans and vector. However, it appears that the consequences differ in the two hosts, with neuropathology in human host, while behavioral changes in the mosquito vector that may be advantageous to the virus.

Dual Anterograde and Retrograde Viral Tracing of Reciprocal Connectivity.

Current large-scale approaches in neuroscience aim to unravel the complete connectivity map of specific neuronal circuits, or even the entire brain. This emerging research discipline has been termed connectomics. Recombinant glycoprotein-deleted rabies virus (RABV ∆G) has become an important tool for the investigation of neuronal connectivity in the brains of a variety of species. Neuronal infection with even a single RABV ∆G particle results in high-level transgene expression, revealing the fine-detailed morphology of all neuronal features-including dendritic spines, axonal processes, and boutons-on a brain-wide scale. This labeling is eminently suitable for subsequent post-hoc morphological analysis, such as semiautomated reconstruction in 3D. Here we describe the use of a recently developed anterograde RABV ∆G variant together with a retrograde RABV ∆G for the investigation of projections both to, and from, a particular brain region. In addition to the automated reconstruction of a dendritic tree, we also give as an example the volume measurements of axonal boutons following RABV ∆G-mediated fluorescent marker expression. In conclusion RABV ∆G variants expressing a combination of markers and/or tools for stimulating/monitoring neuronal activity, used together with genetic or behavioral animal models, promise important insights in the structure-function relationship of neural circuits.

Monosynaptic Tracing in Developing Circuits Using Modified Rabies Virus.

An attenuated rabies virus that expresses fluorescent protein has made it possible to analyze retrograde (presynaptic) monosynaptic connections in vivo. By combining attenuated rabies virus with a Cre-loxP based system to target cells in a subtype-specific fashion, it is possible to examine neuronal input in vivo onto any class of neuron, in development and in the mature brain. We describe here the methods to amplify deletion mutant, pseudotyped rabies virus, selectively target cells of interest using genetic and viral approaches, as well as the stereotaxic procedures required to target neuronal subtypes of interest in vivo.

Epstein-Barr virus exploits host endocytic machinery for cell-to-cell viral transmission rather than a virological synapse.

Epstein-Barr virus (EBV) establishes a lifelong latent infection in B lymphocytes and often is found in epithelial cells. Several lines of evidence indicate that viral transmission mediated by cell-to-cell contact is the dominant mode of infection by EBV for epithelial cells. However, its detailed molecular mechanism has not been fully elucidated. We investigated the role of host membrane trafficking machinery in this process. We have found that adhesion molecules critical for this process are expressed in EBV-positive and -negative Burkitt's lymphoma (BL) cells and multiple epithelial cell lines. Treatment with blocking antibodies against ß1 and ß2 integrin families and their ligands suppressed EBV transmission in a dose-dependent manner. We also confirmed that adhesion molecules are upregulated in co-cultured BL cells. Immunofluorescence staining revealed that the intracellular adhesion molecule 1 (ICAM-1) distributed to the cell surface and partially co-localized with recycling endosomes in co-cultured BL cells. Moreover, cell-to-cell EBV transmission was inhibited upon blocking endocytic recycling by expression of a dominant-negative form of a small GTPase Rab11 or by knockdown of Rab11, supporting the notion that the endocytic pathway-dependent trafficking of ICAM-1 to the cell surface of BL cells contributes to viral transmission by stabilizing cell-to-cell contact between the donor cells and recipient cells. Finally, we demonstrated that co-cultivation upregulated clathrin-mediated endocytosis in the recipient cells, allowing EBV to be internalized. Taken together, our findings demonstrate that EBV exploits host endocytic machinery in both donor and recipient cells, a process which is facilitated by cell-to-cell contact, thereby promoting successful viral transmission.

The neurotrophin receptor p75 mediates gp120-induced loss of synaptic spines in aging mice.

Human immunodeficiency virus 1 and its envelope protein gp120 reduce synaptodendritic complexity. However, the mechanisms contributing to this pathological feature are still not understood. The proneurotrophin brain-derived neurotrophic factor promotes synaptic simplification through the activation of the p75 neurotrophin receptor (p75NTR). Here, we have used gp120 transgenic (gp120tg) mice to investigate whether p75NTR has a role in gp120-mediated neurotoxicity. Old (∼10 months) gp120tg mice exhibited an increase in proneurotrophin brain-derived neurotrophic factor levels in the hippocampus as well as a decrease in the number of dendritic spines when compared to age-matched wild type. These effects were not observed in 3- or 6-month-old mice. To test if the reduction in spine density and morphology is caused by the activation of p75NTR, we crossed gp120tg mice with p75NTR null mice. We found that deletion of only 1 copy of the p75NTR gene in gp120tg mice is sufficient to normalize the number of hippocampal spines, strongly suggesting that the neurotoxic effect of gp120 is mediated by p75NTR. These data indicate that p75NTR antagonists could provide an adjunct therapy against synaptic simplification caused by human immunodeficiency virus 1.

Decreased glial and synaptic glutamate uptake in the striatum of HIV-1 gp120 transgenic mice.

The mechanisms leading to the neurocognitive deficits in humans with immunodeficiency virus type 1 (HIV-1) are not well resolved. A number of cell culture models have demonstrated that the HIV-envelope glycoprotein 120 (gp120) decreases the reuptake of glutamate, which is necessary for learning, memory, and synaptic plasticity. However, the impact of brain HIV-1 gp120 on glutamate uptake systems in vivo remains unknown. Notably, alterations in brain glutamate uptake systems are implicated in a number of neurodegenerative and neurocognitive disorders. We characterized the kinetic properties of system XAG (sodium-dependent) and systems xc- (sodium-independent) [3H]-L-glutamate uptake in the striatum and hippocampus of HIV-1 gp120 transgenic mice, an established model of HIV neuropathology. We determined the kinetic constant Vmax (maximal velocity) and Km (affinity) of both systems XAG and xc- using subcellular preparations derived from neurons and glial cells. We show significant (30-35 %) reductions in the Vmax of systems XAG and xc- in both neuronal and glial preparations derived from the striatum, but not from the hippocampus of gp120 mice relative to wild-type (WT) controls. Moreover, immunoblot analysis showed that the protein expression of glutamate transporter subtype-1 (GLT-1), the predominant brain glutamate transporter, was significantly reduced in the striatum but not in the hippocampus of gp120 mice. These extensive and region-specific deficits of glutamate uptake likely contribute to the development and/or severity of HIV-associated neurocognitive disorders. Understanding the role of striatal glutamate uptake systems in HIV-1 gp120 may advance the development of new therapeutic strategies to prevent neuronal damage and improve cognitive function in HIV patients.

Pseudotyping of G-Gene-Deficient Rabies Virus.

G-deleted fluorescent rabies virus (RV) pseudotyped with RV G proteins, SAD ΔG eGFP (RV CVS-G), can be used as single-round vectors for efficient retrograde labeling of neurons. For these experiments, as well as for monosynaptic tracing, which involves pseudotyping in situ, the use of the CVS strain G is recommended because of its high tropism for neurons. Pseudotype virus stocks generated by transfection of pCAGGS-G (or in MG139-on cells) contain the G protein of the vaccine strain SAD L16, which is broader in its tropism, and infects astrocytes, glia, and oligodendrocytes. We also describe a procedure for pseudotyping with ASLV Env A, which uses a cell-line expressing a version of the EnvA protein that is incorporated efficiently into the RV envelope (EnvARG(RGct)).

Recombinant Fluorescent Rabies Virus Vectors for Tracing Neurons and Synaptic Connections.

Recombinant rabies virus (RV) vectors expressing fluorescent proteins allow staining of neurons from many mammalian species and enable the study of neuron morphology. Because viral spread occurs only between neurons that have synaptic connections, these vectors also permit transsynaptic tracing. A recently established system for restriction of transsynaptic tracing to a single transsynaptic jump, dubbed monosynaptic tracing, uses glycoprotein gene-defective, pseudotyped RV. This allows infection of defined cells and transient complementation with the glycoprotein in situ to support a single step of transsynaptic crossing to presynaptic cells. Here, we introduce protocols describing the production of RV vectors, including the recovery of recombinant RV from complementary DNA (cDNA) and virus pseudotyping in vitro. This allows retrograde staining of neurons projecting to the inoculation site.

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling.

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)(1). PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site(2,3). Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)(4,5). This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3'-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites(6-11).