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.

Immune and Neural Response to Acute Social Stress in Adolescent Humans and Rodents.

Studies in adults have linked stress-related activation of the immune system to the manifestation of psychiatric conditions. Using a translational design, this study aimed to examine the impact of social stress on immune activity in adolescents and on neuronal activity in a preclinical mouse model. Participants were 31 adolescents (ages 12-19), including 25 with mood and anxiety symptoms. Whole-blood samples were collected before and after the Trier Social Stress Test (TSST), a stress-inducing public speaking task, then cultured for 6 hours in the presence and absence of the inflammatory endotoxin lipopolysaccharide (LPS). Effects of TSST and LPS on 41 immune biomarkers were examined using repeated-measures analysis of variance. Separately, juvenile (8-week-old) male mice were non-stressed or exposed to reminder social defeat then intraperitoneally injected with saline or LPS (n = 6/group). Brains were perfused and collected for immunohistochemistry and confocal microscopy at 0, 1, 6, and 24 hours post-injection. Activity was determined by the density of cFos-positive neurons in the paraventricular hypothalamus, paraventricular thalamus, and basolateral amygdala, regions known to show sustained activation to immunological challenge. Analyses in the adolescent study indicated a strong effect of LPS but no effects of TSST or TSST×LPS interaction on immune biomarkers. Similarly, reminder social defeat did not induce sustained neuronal activity changes comparable to LPS immunological challenge in juvenile mice. Our convergent findings across species suggest that the acute immune response to stress documented in adults is not present in youth. Thus, aging and chronicity effects may play an important role in the inflammatory response to acute psychosocial stress.

Astrocyte reactivity and inflammation-induced depression-like behaviors are regulated by Orai1 calcium channels.

Astrocytes contribute to brain inflammation in neurological disorders but the molecular mechanisms controlling astrocyte reactivity and their relationship to neuroinflammatory endpoints are complex and poorly understood. In this study, we assessed the role of the calcium channel, Orai1, for astrocyte reactivity and inflammation-evoked depression behaviors in mice. Transcriptomics and metabolomics analysis indicated that deletion of Orai1 in astrocytes downregulates genes in inflammation and immunity, metabolism, and cell cycle pathways, and reduces cellular metabolites and ATP production. Systemic inflammation by peripheral lipopolysaccharide (LPS) increases hippocampal inflammatory markers in WT but not in astrocyte Orai1 knockout mice. Loss of Orai1 also blunts inflammation-induced astrocyte Ca2+ signaling and inhibitory neurotransmission in the hippocampus. In line with these cellular changes, Orai1 knockout mice showed amelioration of LPS-evoked depression-like behaviors including anhedonia and helplessness. These findings identify Orai1 as an important signaling hub controlling astrocyte reactivity and astrocyte-mediated brain inflammation that is commonly observed in many neurological disorders.

Multivariate Assessment and Risk Ranking of Pesticide Residues in Citrus Fruits.

Pesticides are extensively used in the cultivation and postharvest protection of citrus fruits, therefore continuous monitoring and health risk assessments of their residues are required. This study aimed to investigate the occurrence of pesticide residues on citrus fruits and to evaluate the acute and chronic risk for adults and children. The risk ranking of twenty-three detected pesticides was carried out according to a matrix ranking scheme. Multiple residues were detected in 83% of 76 analyzed samples. In addition, 28% contained pesticides at or above maximum residue levels (MRLs). The most frequently detected pesticides were imazalil, azoxystrobin, and dimethomorph. According to the risk ranking method, imazalil was classified in the high-risk group, followed by prochloraz, chlorpyrifos, azinphos-methyl, tebufenpyrad, and fenpiroximate, which were considered to pose a medium risk. The majority of detected pesticides (74%) posed a low risk. The health risk assessment indicated that imazalil and thiabendazole contribute to acute (HQa) and chronic (HQc) dietary risk, respectively. The HQc was negligible for the general population, while the HQa of imazalil and thiabendazole exceeded the acceptable level in the worst-case scenario. Cumulative chronic/acute risk (HIc/HIa) assessment showed that chronic risk was acceptable in all samples for children and adults, while the acute risk was unacceptable in 5.3% of citrus fruits for adults and 26% of citrus fruits for children. Sensitivity analyses indicated that the ingestion rate and individual body weight were the most influential risk factors.

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.

Stress-induced changes of the cholinergic circuitry promote retrieval-based generalization of aversive memories.

Generalization, the process of applying knowledge acquired in one context to other contexts, often drives the expression of similar behaviors in related situations. At the cellular level, generalization is thought to depend on the activity of overlapping neurons that represent shared features between contexts (general representations). Using contextual fear conditioning in mice, we demonstrate that generalization can also occur in response to stress and result from reactivation of specific, rather than general context representations. We found that generalization emerges during memory retrieval, along with stress-induced abnormalities of septohippocampal oscillatory activity and acetylcholine release, which are typically found in negative affective states. In hippocampal neurons that represent aversive memories and drive generalization, cholinergic septohippocampal afferents contributed to a unique reactivation pattern of cFos, Npas4, and repressor element-1 silencing transcription factor (REST). Together, these findings suggest that generalization can be triggered by perceptually dissimilar but valence-congruent memories of specific aversive experiences. Through promoting the reactivation of such memories and their interference with ongoing behavior, abnormal cholinergic signaling could underlie maladaptive cognitive and behavioral generalization linked to negative affective states.

Microbial fuel cells as an electrical energy source for degradation followed by decolorization of Reactive Black 5 azo dye.

The problem of wastewater has long been ubiquitous and has great consequences for the environment and its inhabitants. Microbial fuel cells (MFCs) have enormous potential for the treatment of wastewaters polluted with azo dyes. The amount of energy that can be produced from a single-chamber MFC is sufficient to perform decolorization and degradation of such dyes, which are widely used in the textile industry. This study on the azo dye, reactive black 5 (RB5), provides an alternative method through three parallel-connected MFCs to obtain electricity that directly serves for the dye's electrochemical degradation. We examined degradation followed by decolorization of RB5 using Fe and Pt electrodes, together with H2O2, to achieve the electro-Fenton process. The amount of voltage produced (295 mV), the current density (276 mA m-3) and the power density (50 mW m-3) were sufficient to degrade 25 mg L-1 RB5 dye with 0.5 mM H2O2 in just 2 h. The dye degradation mechanism was investigated using UV-VIS, FT-IR and HPLC-MS/MS. The ecotoxicity of the degradation products was assessed using a bacterial model, Aliivibrio fischeri. These tests showed that there was successful degradation of the dye to products whose toxicity is less than that of RB5.

From chronic stress and anxiety to neurodegeneration: Focus on neuromodulation of the axon initial segment.

To adapt to the sustained demands of chronic stress, discrete brain circuits undergo structural and functional changes often resulting in anxiety disorders. In some individuals, anxiety disorders precede the development of motor symptoms of Parkinson's disease (PD) caused by degeneration of neurons in the substantia nigra (SN). Here, we present a circuit framework for probing a causal link between chronic stress, anxiety, and PD, which postulates a central role of abnormal neuromodulation of the SN's axon initial segment by brainstem inputs. It is grounded in findings demonstrating that the earliest PD pathologies occur in the stress-responsive, emotion regulation network of the brainstem, which provides the SN with dense aminergic and cholinergic innervation. SN's axon initial segment (AIS) has unique features that support the sustained and bidirectional propagation of activity in response to synaptic inputs. It is therefore, especially sensitive to circuit-mediated stress-induced imbalance of neuromodulation, and thus a plausible initiating site of neurodegeneration. This could explain why, although secondary to pathophysiologies in other brainstem nuclei, SN degeneration is the most extensive. Consequently, the cardinal symptom of PD, severe motor deficits, arise from degeneration of the nigrostriatal pathway rather than other brainstem nuclei. Understanding when and how circuit dysfunctions underlying anxiety can progress to neurodegeneration, raises the prospect of timed interventions for reversing, or at least impeding, the early pathophysiologies that lead to PD and possibly other neurodegenerative disorders.

GluN2A-ERK-mTOR pathway confers a vulnerability to LPS-induced depressive-like behaviour.

Inflammation plays a key role in the pathogenesis of the major depressive disorder. Namely, neuroinflammation can induce the production of neuroactive metabolites that interfere with N-methyl-D-aspartate receptors (NMDAR)-mediated glutamatergic neurotransmission and contribute to depressive-like behaviour. On the other hand, mammalian target of rapamycin (mTOR) activity with synaptogenic effects is the main mediator of antidepressant effects of several potent NMDAR antagonists. In this study, we investigated the specific role of GluN2A subunits of NMDAR on the activity of mTOR signaling and behaviour in lipopolysaccharide (LPS)-induces model of depression. The results showed that mice lacking GluN2A subunit did not display depressive-like behavior after the immune challenge, opposite to LPS-treated wild-type mice. Specifically, in GluN2A knockout mice, we estimated the activity of the mTOR pathway in the hippocampus and prefrontal cortex (PFC) by measuring synaptic levels of upstream regulators (p-Akt, p-ERK, and p-GSK3ß) and downstream effectors (p-mTOR, and p-p70S6K) of mTOR activity. In addition, we assessed the changes in the levels of two important synaptic markers, GluA1 and PSD-95. Contrary to downregulated mTOR signaling and decreased synaptic markers in LPS-treated wild-type animals, the resilience of GluN2A KO mice to depressive-like behaviour was paralleled with sustained mTOR signaling activity synaptic stability in hippocampus and PFC. Finally, we disclosed that resistance of GluN2A knockouts to LPS-induced depressive-like behavior was ERK-dependent. These findings demonstrate that GluN2A-ERK-mTOR signaling is a vulnerability factor of inflammation-related depressive behaviour, making this signaling pathway the promising target for developing novel antidepressants.

Stress-induced generalization of negative memories is mediated by an extended hippocampal circuit.

Memories of negative experiences exert important control of behavior in the face of actual or anticipated threat. Sometimes, however, this control extends to non-threatening situations, a phenomenon known as overgeneralization of negative memories. Overgeneralization is a reliable cognitive phenotype of major depressive disorder, generalized anxiety disorder, and post-traumatic stress disorder. We therefore sought to develop an animal model to study stress-induced generalization of negative memories (SIG) and determine its dependence on the episodic-like memory circuit. We found that male and female mice, which were trained to differentiate a threatening from neutral context, exhibited robust SIG in response to subsequent social stress. Using chemogenetic circuit manipulations during memory retrieval, we demonstrated that both excitatory afferents to the dorsal hippocampus (DH) from the ventral tegmental area (VTA), and excitatory efferents from the DH to the retrosplenial cortex (RSC) contribute to SIG. Based on the known roles of these projections, we suggest that (1) by targeting subcortical VTA circuits that provide valence signals to the DH, stress prioritizes the retrieval of negative over neutral memories, and (2) by forwarding such information to the RSC, stress engages cortical mechanisms that support the retrieval of general relative to specific memory features. Altogether, these results suggest that various components of the extended hippocampal circuit can serve as treatment targets for memory overgeneralization.

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).

PFC mTOR signaling as a biological signature for cognitive deficits in bipolar disorder without psychosis.

Vanderplow et al.1 report decreased PFC Akt-mTOR signaling in males with bipolar disorder (BD) without psychosis compared with those with psychosis, possibly related to cognitive deficits. Understanding how cognition differs between these BD subtypes clinically and biologically remains a challenge.

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.

Activation of the dorsal, but not the ventral, hippocampus relieves neuropathic pain in rodents.

ABSTRACT: Accumulating evidence suggests hippocampal impairment under the chronic pain phenotype. However, it is unknown whether neuropathic behaviors are related to dysfunction of the hippocampal circuitry. Here, we enhanced hippocampal activity by pharmacological, optogenetic, and chemogenetic techniques to determine hippocampal influence on neuropathic pain behaviors. We found that excitation of the dorsal (DH), but not the ventral (VH) hippocampus induces analgesia in 2 rodent models of neuropathic pain (SNI and SNL) and in rats and mice. Optogenetic and pharmacological manipulations of DH neurons demonstrated that DH-induced analgesia was mediated by N-Methyl-D-aspartate and µ-opioid receptors. In addition to analgesia, optogenetic stimulation of the DH in SNI mice also resulted in enhanced real-time conditioned place preference for the chamber where the DH was activated, a finding consistent with pain relief. Similar manipulations in the VH were ineffective. Using chemo-functional magnetic resonance imaging (fMRI), where awake resting-state fMRI was combined with viral vector-mediated chemogenetic activation (PSAM/PSEM89s) of DH neurons, we demonstrated changes of functional connectivity between the DH and thalamus and somatosensory regions that tracked the extent of relief from tactile allodynia. Moreover, we examined hippocampal functional connectivity in humans and observe differential reorganization of its anterior and posterior subdivisions between subacute and chronic back pain. Altogether, these results imply that downregulation of the DH circuitry during chronic neuropathic pain aggravates pain-related behaviors. Conversely, activation of the DH reverses pain-related behaviors through local excitatory and opioidergic mechanisms affecting DH functional connectivity. Thus, this study exhibits a novel causal role for the DH but not the VH in controlling neuropathic pain-related behaviors.

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.