Formation of memory assemblies through the DNA-sensing TLR9 pathway.

As hippocampal neurons respond to diverse types of information1, a subset assembles into microcircuits representing a memory2. Those neurons typically undergo energy-intensive molecular adaptations, occasionally resulting in transient DNA damage3-5. Here we found discrete clusters of excitatory hippocampal CA1 neurons with persistent double-stranded DNA (dsDNA) breaks, nuclear envelope ruptures and perinuclear release of histone and dsDNA fragments hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of TLR9 signalling and accumulation of centrosomal DNA damage repair complexes6. Neuron-specific knockdown of Tlr9 impaired memory while blunting contextual fear conditioning-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Notably, TLR9 had an essential role in centrosome function, including DNA damage repair, ciliogenesis and build-up of perineuronal nets. We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and TLR9-mediated repair, resulting in their recruitment to memory circuits. With compromised TLR9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments implicated in accelerated senescence, psychiatric disorders and neurodegenerative disorders. Maintaining the integrity of TLR9 inflammatory signalling thus emerges as a promising preventive strategy for neurocognitive deficits.

Sex-specific roles of hippocampal microRNAs in stress vulnerability and resilience.

Contrary to intuition, most individuals are resilient to psychological trauma and only a minority is vulnerable. Men and women are known to respond differently to trauma exposure, however, mechanisms underlying the relationship between sex differences and trauma resilience and vulnerability are not yet fully understood. Taking advantage of the Behavioral Profiling approach, which enables differentiating between 'affected' and 'unaffected' individuals, we examined sex-associated differences in stress exposure effects on hippocampal expression of selected stress-related GABA-A receptor targeting miRNAs. Levels of the miRNA-144 and miRNA-33 were measured in male and female affected (vulnerable, e.g., higher freezing time) and unaffected (resilient) rats. In male rats, increased levels of miRNA-144 and miRNA-33 were observed in the dorsal dentate gyrus (dDG) and ventral dentate gyrus (vDG) respectively, of stress-exposed but unaffected animals. In females, we observed an increased expression of miRNA-144 and miRNA-33 in the ventral cornu ammonis 1 (vCA1) of affected animals. Accordingly, we inhibited miRNAs expression selectively in hippocampal subregions using oligonucleotides containing locked nucleic acid bases, to examine the miRNAs' causal contribution to either vulnerability or resilience to stress in each sex. Inhibition of miRNA-144 in dDG and miRNA-33 in vDG in males resulted in an increased prevalence of vulnerable animals, while inhibition of miRNA-144 and miRNA-33 in vCA1 in females increased the proportion of resilient animals. The current findings reveal a critical sex-associated difference in the role of miRNAs in stress vulnerability and resilience. This novel understanding of sex-associated epigenetic involvement in the mechanism of stress-related psychopathologies may help improve gender-specific diagnosis and effective treatment.

Protocol for assessing the role of hippocampal perineuronal nets in aversive memories.

Perineuronal nets (PNNs) are emerging as critical regulators of memory-related neuronal processes. However, their exact contribution depends on type of memory, consolidation stage, or brain region, and remains to be fully investigated. We describe here a protocol to evaluate the importance of PNNs in the dorsal hippocampus in different stages of aversive memories using a mouse model. The protocol provides detailed instructions for surgical implantation of hippocampal cannulas, drug infusion, contextual fear conditioning procedures, and immunohistochemistry for PNN visualization. For complete details on the use and execution of this protocol, please refer to Jovasevic et al. (2021).

Herpesviruses assimilate kinesin to produce motorized viral particles.

Neurotropic alphaherpesviruses initiate infection in exposed mucosal tissues and, unlike most viruses, spread rapidly to sensory and autonomic nerves where life-long latency is established1. Recurrent infections arise sporadically from the peripheral nervous system throughout the life of the host, and invasion of the central nervous system may occur, with severe outcomes2. These viruses directly recruit cellular motors for transport along microtubules in nerve axons, but how the motors are manipulated to deliver the virus to neuronal nuclei is not understood. Here, using herpes simplex virus type I and pseudorabies virus as model alphaherpesviruses, we show that a cellular kinesin motor is captured by virions in epithelial cells, carried between cells, and subsequently used in neurons to traffic to nuclei. Viruses assembled in the absence of kinesin are not neuroinvasive. The findings explain a critical component of the alphaherpesvirus neuroinvasive mechanism and demonstrate that these viruses assimilate a cellular protein as an essential proviral structural component. This principle of viral assimilation may prove relevant to other virus families and offers new strategies to combat infection.

Primary cilia are required for the persistence of memory and stabilization of perineuronal nets.

It is well established that the formation of episodic memories requires multiple hippocampal mechanisms operating on different time scales. Early mechanisms of memory formation (synaptic consolidation) have been extensively characterized. However, delayed mechanisms, which maintain hippocampal activity as memories stabilize in cortical circuits, are not well understood. Here we demonstrate that contrary to the transient expression of early- and delayed-response genes, the expression of cytoskeleton- and extracellular matrix-associated genes remains dynamic even at remote time points. The most profound expression changes clustered around primary cilium-associated and collagen genes. These genes most likely contribute to memory by stabilizing perineuronal nets in the dorsohippocampal CA1 subfield, as revealed by targeted disruptions of the primary cilium or perineuronal nets. The findings show that nonsynaptic, primary cilium-mediated mechanisms are required for the persistence of context memory.

High ethanol preference and dissociated memory are co-occurring phenotypes associated with hippocampal GABAAR-δ receptor levels.

Alcohol use disorder (AUD) frequently co-occurs with dissociative disorders and disorders with dissociative symptoms, suggesting a common neurobiological basis. It has been proposed that facilitated information processing under the influence of alcohol, resulting in the formation of dissociated memories, might be an important factor controlling alcohol use. Access to such memories is facilitated under the effect of alcohol, thus further reinforcing alcohol use. To interrogate possible mechanisms associated with these phenotypes, we used a mouse model of dissociative amnesia, combined with a high-alcohol preferring (HAP) model of AUD. Dissociated memory was induced by activation of hippocampal extrasynaptic GABA type A receptor delta subunits (GABAAR-δ), which control tonic inhibition and to which ethanol binds with high affinity. Increased ethanol preference was associated with increased propensity to form dissociated memories dependent on GABAAR-δ in the dorsal hippocampus (DH). Furthermore, the DH level of GABAAR-δ protein, but not mRNA, was increased in HAP mice, and was inversely correlated to the level of miR-365-3p, suggesting an miRNA-mediated post-transcriptional mechanism contributing to elevated GABAAR-δ. The observed changes of DH GABAAR-δ were associated with a severe reduction of excitatory projections stemming from GABAAR-δ-containing pyramidal neurons in the subiculum and terminating in the mammillary body. These results suggest that both molecular and circuit dysfunction involving hippocampal GABAAR-δ receptors might contribute to the co-occurrence of ethanol preference and dissociated information processing.

Excitatory VTA to DH projections provide a valence signal to memory circuits.

The positive or negative value (valence) of past experiences is normally integrated into neuronal circuits that encode episodic memories and plays an important role in guiding behavior. Here, we show, using mouse behavioral models, that glutamatergic afferents from the ventral tegmental area to the dorsal hippocampus (VTA→DH) signal negative valence to memory circuits, leading to the formation of fear-inducing context memories and to context-specific reinstatement of fear. To a lesser extent, these projections also contributed to opioid-induced place preference, suggesting a role in signaling positive valence as well, and thus a lack of dedicated polarity. Manipulations of VTA terminal activity were more effective in females and paralleled by sex differences in glutamatergic signaling. By prioritizing retrieval of negative and positive over neutral memories, the VTA→DH circuit can facilitate the selection of adaptive behaviors when current and past experiences are valence congruent.

TACC3 Regulates Microtubule Plus-End Dynamics and Cargo Transport in Interphase Cells.

End-binding proteins (EBs) are widely viewed as master regulators of microtubule dynamics and function. Here, we show that while EB1 mediates the dynamic microtubule capture of herpes simplex virus type 1 (HSV-1) in fibroblasts, in neuronal cells, infection occurs independently of EBs through stable microtubules. Prompted by this, we find that transforming acid coiled-coil protein 3 (TACC3), widely studied in mitotic spindle formation, regulates the cytoplasmic localization of the microtubule polymerizing factor chTOG and influences microtubule plus-end dynamics during interphase to control infection in distinct cell types. Furthermore, perturbing TACC3 function in neuronal cells resulted in the formation of disorganized stable, detyrosinated microtubule networks and changes in cellular morphology, as well as impaired trafficking of both HSV-1 and transferrin. These trafficking defects in TACC3-depleted cells were reversed by the depletion of kinesin-1 heavy chains. As such, TACC3 is a critical regulator of interphase microtubule dynamics and stability that influences kinesin-1-based cargo trafficking.

Author Correction: HIV-1 counteracts an innate restriction by amyloid precursor protein resulting in neurodegeneration.

The original version of this Article contained an error in the Methods section 'Viruses and drugs'. The timing for drug treatment of CHME3 4 × 4 or 293T cells with γ-secretase inhibitor or BACE1 inhibitor was incorrectly given as '1 day prior to infection or transfection' and should have stated '4 or 6 h post transfection or infection, respectively'. This error is now corrected in both the PDF and HTML versions of the Article.

HIV-1 counteracts an innate restriction by amyloid precursor protein resulting in neurodegeneration.

While beta-amyloid (Aß), a classic hallmark of Alzheimer's disease (AD) and dementia, has long been known to be elevated in the human immunodeficiency virus type 1 (HIV-1)-infected brain, why and how Aß is produced, along with its contribution to HIV-associated neurocognitive disorder (HAND) remains ill-defined. Here, we reveal that the membrane-associated amyloid precursor protein (APP) is highly expressed in macrophages and microglia, and acts as an innate restriction against HIV-1. APP binds the HIV-1 Gag polyprotein, retains it in lipid rafts and blocks HIV-1 virion production and spread. To escape this restriction, Gag promotes secretase-dependent cleavage of APP, resulting in the overproduction of toxic Aß isoforms. This Gag-mediated Aß production results in increased degeneration of primary cortical neurons, and can be prevented by γ-secretase inhibitor treatment. Interfering with HIV-1's evasion of APP-mediated restriction also suppresses HIV-1 spread, offering a potential strategy to both treat infection and prevent HAND.

Neurobiological mechanisms of state-dependent learning.

State-dependent learning (SDL) is a phenomenon relating to information storage and retrieval restricted to discrete states. While extensively studied using psychopharmacological approaches, SDL has not been subjected to rigorous neuroscientific study. Here we present an overview of approaches historically used to induce SDL, and highlight some of the known neurobiological mechanisms, in particular those related to inhibitory neurotransmission and its regulation by microRNAs (miR). We also propose novel cellular and circuit mechanisms as contributing factors. Lastly, we discuss the implications of advancing our knowledge on SDL, both for most fundamental processes of learning and memory as well as for development and maintenance of psychopathology.

Muscarinic acetylcholine receptors act in synergy to facilitate learning and memory.

Understanding how episodic memories are formed and retrieved is necessary if we are to treat disorders in which they malfunction. Muscarinic acetylcholine receptors (mAChR) in the hippocampus and cortex underlie memory formation, but there is conflicting evidence regarding their role in memory retrieval. Additionally, there is no consensus on which mAChR subtypes are critical for memory processing. Using pharmacological and genetic approaches, we found that (1) encoding and retrieval of contextual memory requires mAChR in the dorsal hippocampus (DH) and retrosplenial cortex (RSC), (2) memory formation requires hippocampal M3 and cooperative activity of RSC M1 and M3, and (3) memory retrieval is more impaired by inactivation of multiple M1-M4 mAChR in DH or RSC than inactivation of individual receptor subtypes. Contrary to the view that acetylcholine supports learning but is detrimental to memory retrieval, we found that coactivation of multiple mAChR is required for retrieval of both recently and remotely acquired context memories. Manipulations with higher receptor specificity were generally less potent than manipulations targeting multiple receptor subtypes, suggesting that mAChR act in synergy to regulate memory processes. These findings provide unique insight into the development of therapies for amnestic symptoms, suggesting that broadly acting, rather than receptor-specific, mAchR agonists and positive allosteric modulators may be the most effective therapeutic approach.

Microtubule plus end-associated CLIP-170 initiates HSV-1 retrograde transport in primary human cells.

Dynamic microtubules (MTs) continuously explore the intracellular environment and, through specialized plus end-tracking proteins (+TIPs), engage a variety of targets. However, the nature of cargoes that require +TIP-mediated capture for their movement on MTs remains poorly understood. Using RNA interference and dominant-negative approaches, combined with live cell imaging, we show that herpes simplex virus particles that have entered primary human cells exploit a +TIP complex comprising end-binding protein 1 (EB1), cytoplasmic linker protein 170 (CLIP-170), and dynactin-1 (DCTN1) to initiate retrograde transport. Depletion of these +TIPs completely blocked post-entry long-range transport of virus particles and suppressed infection ∼5,000-fold, whereas transferrin uptake, early endosome organization, and dynein-dependent movement of lysosomes and mitochondria remained unaffected. These findings provide the first insights into the earliest stages of viral engagement of MTs through specific +TIPs, akin to receptors, with therapeutic implications, and identify herpesvirus particles as one of a very limited number of cargoes absolutely dependent on CLIP-170-mediated capture to initiate transport in primary human cells.

GABAergic mechanisms regulated by miR-33 encode state-dependent fear.

Fear-inducing memories can be state dependent, meaning that they can best be retrieved if the brain states at encoding and retrieval are similar. Restricted access to such memories can present a risk for psychiatric disorders and hamper their treatment. To better understand the mechanisms underlying state-dependent fear, we used a mouse model of contextual fear conditioning. We found that heightened activity of hippocampal extrasynaptic GABAA receptors, believed to impair fear and memory, actually enabled their state-dependent encoding and retrieval. This effect required protein kinase C-ßII and was influenced by miR-33, a microRNA that regulates several GABA-related proteins. In the extended hippocampal circuit, extrasynaptic GABAA receptors promoted subcortical, but impaired cortical, activation during memory encoding of context fear. Moreover, suppression of retrosplenial cortical activity, which normally impairs retrieval, had an enhancing effect on the retrieval of state-dependent fear. These mechanisms can serve as treatment targets for managing access to state-dependent memories of stressful experiences.

Double Dissociation of the Roles of Metabotropic Glutamate Receptor 5 and Oxytocin Receptor in Discrete Social Behaviors.

Social interactions in vertebrates are complex phenomena based on affective and cognitive processes. Multiple brain regions and neurotransmitter systems are involved in the expression of social behaviors, but their individual roles in specific aspects of social interactions are not well understood. Here we investigated how Gq-protein-coupled metabotropic glutamate receptor 5 (mGluR5) and oxytocin receptor (Oxtr) affect social affiliation and social memory. We used conditional genetic approaches in which the genes coding for these receptors were knocked out in the lateral septum by infusion of recombinant adeno-associated viral vectors containing Cre recombinase (AAV-Cre). Social behavior was assessed 2 weeks later using a three-chamber paradigm for sociability and preference for social novelty. Septal deletion of mGluR5 abolished sociability while leaving preference for social novelty intact. In contrast, deletion of Oxtr did not affect sociability but significantly impaired preference for social novelty. Nonsocial behaviors or memories, including novel object recognition or fear conditioning, were not affected by these genetic manipulations. Immunohistochemical analyses of the distribution of mGluR5 and Oxtr revealed non-overlapping localization of these receptors within the lateral septum, suggesting that not only different neurotransmitters but also different neuronal types contribute to sociability versus preference for social novelty. Our findings identify highly specialized roles of lateral septal mGluR5 and Oxtr in the the regulation of discrete social behaviors, and suggest that deficits in social interactions, which accompany many mental illnesses, would benefit from comprehensive treatments targeting different components of social functioning.

HIV-1 capsids bind and exploit the kinesin-1 adaptor FEZ1 for inward movement to the nucleus.

Intracellular transport of cargos, including many viruses, involves directed movement on microtubules mediated by motor proteins. Although a number of viruses bind motors of opposing directionality, how they associate with and control these motors to accomplish directed movement remains poorly understood. Here we show that human immunodeficiency virus type 1 (HIV-1) associates with the kinesin-1 adaptor protein, Fasiculation and Elongation Factor zeta 1 (FEZ1). RNAi-mediated FEZ1 depletion blocks early infection, with virus particles exhibiting bi-directional motility but no net movement to the nucleus. Furthermore, both dynein and kinesin-1 motors are required for HIV-1 trafficking to the nucleus. Finally, the ability of exogenously expressed FEZ1 to promote early HIV-1 infection requires binding to kinesin-1. Our findings demonstrate that opposing motors both contribute to early HIV-1 movement and identify the kinesin-1 adaptor, FEZ1 as a capsid-associated host regulator of this process usurped by HIV-1 to accomplish net inward movement towards the nucleus.

Fear-enhancing effects of septal oxytocin receptors.

The nonapeptide oxytocin is considered beneficial to mental health due to its anxiolytic, prosocial and antistress effects, but evidence for anxiogenic actions of oxytocin in humans has recently emerged. Using region-specific manipulations of the mouse oxytocin receptor (Oxtr) gene (Oxtr), we identified the lateral septum as the brain region mediating fear-enhancing effects of Oxtr. These effects emerge after social defeat and require Oxtr specifically coupled to the extracellular signal-regulated protein kinase pathway.

Fear conditioning and extinction: emotional states encoded by distinct signaling pathways.

Conditioning and extinction of fear have traditionally been viewed as two independent learning processes for encoding representations of contexts or cues (conditioned stimuli, CS), aversive events (unconditioned stimuli, US), and their relationship. Based on the analysis of protein kinase signaling patterns in neurons of the fear circuit, we propose that fear and extinction are best conceptualized as emotional states triggered by a single CS representation with two opposing values: aversive and non-aversive. These values are conferred by the presence or absence of the US and encoded by distinct sets of kinase signaling pathways and their downstream targets. Modulating specific protein kinases thus has the potential to modify emotional states, and hence, may emerge as a promising treatment for anxiety disorders.

NMDA receptors in retrosplenial cortex are necessary for retrieval of recent and remote context fear memory.

Over time, memory retrieval is thought to transfer from the hippocampus to a distributed network of neocortical sites. Of these sites, the retrosplenial cortex (RSC) is robustly activated during retrieval of remotely acquired, emotionally valenced memories. It is unclear, however, whether RSC is specifically involved in memory storage or retrieval, and which neurotransmitter receptor mechanisms serve its function. We addressed these questions by inhibiting NMDARs in RSC via infusions of APV before tests for context fear in mice. Anterior cingulate cortex (ACC) and dorsal hippocampus (DH), which have been implicated in the retrieval of remote and recent memory, respectively, served as neuroanatomical controls. Surprisingly, infusion of APV only into RSC, but not ACC or DH, abolished retrieval of remote memory, as revealed by lack of freezing to the conditioning context. APV infused into RSC also impaired retrieval of recent memory, but had no effect on conditioning or memory storage. Within-subject experiments confirmed that the role of RSC in memory retrieval is not time limited. RSC-dependent context fear memory retrieval was mediated by NR2A, but not NR2B, subunit-containing NMDARs. Collectively, these data are the first demonstration that NMDARs in RSC are necessary for the retrieval of remote and recent memories of fear-evoking contexts. Dysfunction of RSC may thereby contribute significantly to the reexperiencing of traumatic memories in patients with posttraumatic stress disorder.