The basolateral amygdala (BLA) and ventral hippocampal CA1 (vCA1) are cellularly and functionally diverse along their anterior–posterior and superficial-deep axes. Here, we find that anterior BLA (aBLA) and posterior BLA (pBLA) innervate deep-layer calbindin1-negative (Calb1−) and superficial-layer calbindin1-positive neurons (Calb1+) in vCA1, respectively. Photostimulation of pBLA–vCA1 inputs has an anxiolytic effect in mice, promoting approach behaviours during conflict exploratory tasks. By contrast, stimulating aBLA–vCA1 inputs induces anxiety-like behaviour resulting in fewer approaches. During conflict stages of the elevated plus maze task vCA1^Calb1+ neurons are preferentially activated at the open-to-closed arm transition, and photostimulation of vCA1^Calb1+ neurons at decision-making zones promotes approach with fewer retreats. In the APP/PS1 mouse model of Alzheimer’s disease, which shows anxiety-like behaviour, photostimulating the pBLA–vCA1^Calb1+ circuit ameliorates the anxiety in a Calb1-dependent manner. These findings suggest the pBLA–vCA1^Calb1+ circuit from heterogeneous BLA–vCA1 connections drives approach behaviour to reduce anxiety-like behaviour.

Herpes simplex virus type 1 strain 129 (H129) has represented a promising anterograde neuronal circuit tracing tool, which complements the existing retrograde tracers. However, the current H129 derived tracers are multisynaptic, neither bright enough to label the details of neurons nor capable of determining direct projection targets as monosynaptic tracer. Based on the bacterial artificial chromosome of H129, we have generated a serial of recombinant viruses for neuronal circuit tracing. Among them, H129-G4 was obtained by inserting binary tandemly connected GFP cassettes into the H129 genome, and H129-ΔTK-tdT was obtained by deleting the thymidine kinase (TK) gene and adding tdTomato coding gene to the H129 genome. Then the obtained viral tracers were tested in vitro and in vivo for the tracing capacity. H129-G4 is capable of transmitting through multiple synapses, labeling the neurons by green florescent protein, and visualizing the morphological details of the labeled neurons. H129-ΔTK-tdT neither replicates nor spreads in neurons alone, but transmits to and labels the postsynaptic neurons with tdTomato in the presence of complementary expressed TK from a helper virus. H129-ΔTK-tdT is also capable to map the direct projectome of the specific neuron type in the given brain regions in Cre transgenic mice. In the tested brain regions where circuits are well known, the H129-ΔTK-tdT tracing patterns are consistent with the previous results. With the assistance of the helper virus complimentarily expressing TK, H129-ΔTK-tdT replicates in the initially infected neuron, transmits anterogradely through one synapse, and labeled the postsynaptic neurons with tdTomato. The H129-ΔTK-tdT anterograde monosynaptic tracing system offers a useful tool for mapping the direct output in neuronal circuitry. H129-G4 is an anterograde multisynaptic tracer with a labeling signal strong enough to display the details of neuron morphology.

Congenital human cytomegalovirus (HCMV) infection is the leading cause of neurological disabilities in children worldwide, but the mechanisms underlying these disorders are far from well-defined. HCMV infection has been shown to dysregulate the Notch signaling pathway in human neural progenitor cells (NPCs). As an important downstream effector of Notch signaling, the transcriptional regulator Hairy and Enhancer of Split 1 (Hes1) is essential for governing NPC fate and fetal brain development. In the present study, we report that HCMV infection downregulates Hes1 protein levels in infected NPCs. The HCMV 72-kDa immediate-early 1 protein (IE1) is involved in Hes1 degradation by assembling a ubiquitination complex and promoting Hes1 ubiquitination as a potential E3 ubiquitin ligase, followed by proteasomal degradation of Hes1. Sp100A, an important component of PML nuclear bodies, is identified to be another target of IE1-mediated ubiquitination. A C-terminal acidic region in IE1, spanning amino acids 451 to 475, is required for IE1/Hes1 physical interaction and IE1-mediated Hes1 ubiquitination, but is dispensable for IE1/Sp100A interaction and ubiquitination. Our study suggests a novel mechanism linking downregulation of Hes1 protein to neurodevelopmental disorders caused by HCMV infection. Our findings also complement the current knowledge of herpesviruses by identifying IE1 as the first potential HCMV-encoded E3 ubiquitin ligase. Congenital human cytomegalovirus (HCMV) infection is the leading cause of neurological disabilities in children, but the underlying pathogenesis of this infection remains unclear. Hes1, an important effector of Notch signaling, governs the fate of neural progenitor cells (NPCs) and fetal brain development. Here we demonstrate that: (1) HCMV infection results in loss of Hes1 protein in NPCs; (2) the HCMV immediate-early 1 protein (IE1) mediates Hes1 protein downregulation through direct interaction, which requires amino acids 451–475; (3) IE1 assembles a Hes1 ubiquitination complex and mediates Hes1 ubiquitination; and (4) IE1 also assembles an Sp100A ubiquitination complex and mediates Sp100A ubiquitination, but does not require amino acids 451–475. These results suggest that HCMV IE1 is a potential E3 ubiquitin ligase. Downregulation of Hes1 by HCMV infection and IE1 implies a novel mechanism linking Hes1 depletion to virus-induced neuropathogenesis.

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neurodevelopmental disorders. However, the neuropathogenesis remains largely elusive due to a lack of informative animal models. In this study, we developed a congenital murine CMV (cMCMV) infection mouse model with high survival rate and long survival period that allowed long-term follow-up study of neurodevelopmental disorders. This model involves in utero intracranial injection and mimics many reported clinical manifestations of cCMV infection in infants, including growth restriction, hearing loss, and impaired cognitive and learning-memory abilities. We observed that abnormalities in MRI/CT neuroimaging were consistent with brain hemorrhage and loss of brain parenchyma, which was confirmed by pathological analysis. Neuropathological findings included ventriculomegaly and cortical atrophy associated with impaired proliferation and migration of neural progenitor cells in the developing brain at both embryonic and postnatal stages. Robust inflammatory responses during infection were shown by elevated inflammatory cytokine levels, leukocyte infiltration, and activation of microglia and astrocytes in the brain. Pathological analyses and CT neuroimaging revealed brain calcifications induced by cMCMV infection and cell death via pyroptosis. Furthermore, antiviral treatment with ganciclovir significantly improved neurological functions and mitigated brain damage as shown by CT neuroimaging. These results demonstrate that this model is suitable for investigation of mechanisms of infection-induced brain damage and long-term studies of neurodevelopmental disorders, including the development of interventions to limit CNS damage associated with cCMV infection.

Monosynaptic viral tracers are essential tools for dissecting neuronal connectomes and for targeted delivery of molecular sensors and effectors. Viral toxicity and complex multi-injection protocols are major limiting application barriers. To overcome these barriers, we developed an anterograde monosynaptic H129_Amp tracer system based on HSV-1 strain H129. The H129_Amp tracer system consists of two components: an H129-dTK-T2-pac^Flox helper which assists H129_Amp tracer’s propagation and transneuronal monosynaptic transmission. The shared viral features of tracer/helper allow for simultaneous single-injection and subsequent high expression efficiency from multiple-copy of expression cassettes in H129_Amp tracer. These improvements of H129_Amp tracer system shorten experiment duration from 28-day to 5-day for fast-bright monosynaptic tracing. The lack of toxic viral genes in the H129_Amp tracer minimizes toxicity in postsynaptic neurons, thus offering the potential for functional anterograde mapping and long-term tracer delivery of genetic payloads. The H129_Amp tracer system is a powerful tracing tool for revealing neuronal connectomes.

Mediator of IRF3 activation ([MITA] also known as STING) is a direct sensor of cyclic dinucleotide and critically mediates cytoplasmic DNA­–triggered innate immune signaling. The activity of MITA is extensively regulated by ubiquitination and deubiquitination. In this study, we report that USP20 interacts with and removes K48-linked ubiquitin chains from MITA after HSV-1 infection, thereby stabilizing MITA and promoting cellular antiviral responses. Deletion of USP20 accelerates HSV-1–induced degradation of MITA and impairs phosphorylation of IRF3 and IκBα as well as subsequent induction of type I IFNs and proinflammatory cytokines after HSV-1 infection or cytoplasmic DNA challenge. Consistently, Usp20^−/− mice produce decreased type I IFNs and proinflammatory cytokines, exhibit increased susceptibility to lethal HSV-1 infection, and aggravated HSV-1 replication compared with Usp20^+/+ mice. In addition, complement of MITA into Usp20^−/− cells fully restores HSV-1–triggered signaling and inhibits HSV-1 infection. These findings suggest a crucial role of USP20 in maintaining the stability of MITA and promoting innate antiviral signaling. Key Points Knockout of USP20 inhibits HSV-1– but not Sendai virus–triggered signaling. USP20-deficient mice exhibit increased susceptibility to HSV-1 infection. USP20 deconjugates K48-linked polyubiquitin chains from and thereby stabilizes MITA.