C-fos Expression Research Articles

Metformin attenuates central sensitization by regulating neuroinflammation through the TREM2-SYK signaling pathway in a mouse model of chronic migraine

BackgroundChronic migraine (CM) is a serious neurological disorder. Central sensitization is one of the important pathophysiological mechanisms underlying CM, and microglia-induced neuroinflammation conduces to central sensitization. Triggering receptor expressed on myeloid cells 2 (TREM2) is presented solely in microglia residing within the central nervous system and plays a key role in neuroinflammation. Metformin has been shown to regulate inflammatory responses and exert analgesic effects, but its relationship with CM remains unclear. In the study, we investigated whether metformin modulates TREM2 to improve central sensitization of CM and clarified the potential molecular mechanisms.MethodsA CM mouse model was induced by administration of nitroglycerin (NTG). Behavioral evaluations were conducted using von Frey filaments and hot plate experiments. Western blot and immunofluorescence techniques were employed to investigate the molecular mechanisms. Metformin and the SYK inhibitor R406 were administered to mice to assess their regulatory effects on neuroinflammation and central sensitization. To explore the role of TREM2-SYK in regulating neuroinflammation with metformin, a lentivirus encoding TREM2 was injected into the trigeminal nucleus caudalis (TNC). In vitro experiments were conducted to evaluate the regulation of TREM2-SYK by metformin, involving interventions with LPS, metformin, R406, siTREM2, and TREM2 plasmids.ResultsMetformin and R406 pretreatment can effectively improve hyperalgesia in CM mice. Both metformin and R406 significantly inhibit c-fos and CGRP expression in CM mice, effectively suppressing the activation of microglia and NLRP3 inflammasome induced by NTG. With the administration of NTG, TREM2 expression gradually increased in TNC microglia. Additionally, we observed that metformin significantly inhibits TREM2 and SYK expression in CM mice. Lv-TREM2 attenuated metformin-mediated anti-inflammatory responses. In vitro experiments, knockdown of TREM2 inhibited LPS-induced SYK pathway activation and alleviated inflammatory responses. After the sole overexpression of TREM2, the SYK signaling pathway is activated, resulting in the activation of the NLRP3 inflammasome and an increased expression of pro-inflammatory cytokines; nevertheless, this consequence can be reversed by R406. The overexpression of TREM2 attenuates the inhibition of SYK activity mediated by metformin, and this effect can be reversed by R406.ConclusionsOur findings suggest that metformin attenuates central sensitization in CM by regulating the activation of microglia and NLRP3 inflammasome through the TREM2-SYK pathway.Graphical

Wireless optogenetic stimulation on the prelimbic to the nucleus accumbens core circuit attenuates cocaine-induced behavioral sensitization

Behavioral sensitization is defined as the heightened and persistent behavioral response to repeated drug exposure as a manifestation of drug craving. Psychomotor stimulants such as cocaine can induce strong behavioral sensitization. In this study, we explored the effects of optogenetic stimulation of the prelimbic (PL) to the nucleus accumbnes (NAc) core on the expression of cocaine-induced behavioral sensitization. Using wireless optogenetics, we selectively stimulated the PL-NAc core circuit, and assessed the effects of this treatment on cocaine-induced locomotor activity and accompanying changes in neuronal activation and dendritic spine density. Our findings revealed that optogenetic stimulation of the PL-NAc core circuit effectively suppressed the cocaine-induced locomotor sensitization, accompanied by a reduction in c-Fos expression within the NAc core. Moreover, optogenetic stimulation led to reduction in dendritic spine density, particularly thin and mushroom spine densities, in the NAc core. This study demonstrates that cocaine-induced locomotor sensitization can be regulated by optogenetic stimulation of the PL-NAc core circuit, providing insights into the crucial role of this circuit in psychomotor stimulant addiction.

Dorsal raphe to basolateral amygdala corticotropin-releasing factor circuit regulates cocaine-memory reconsolidation.

Environmental stimuli elicit drug craving and relapse in cocaine users by triggering the retrieval of strong cocaine-related contextual memories. Retrieval can also destabilize drug memories, requiring reconsolidation, a protein synthesis-dependent storage process, to maintain memory strength. Corticotropin-releasing factor (CRF) signaling in the basolateral amygdala (BLA) is necessary for cocaine-memory reconsolidation. We have hypothesized that a critical source of CRF in the BLA is the dorsal raphe nucleus (DR) based on its neurochemistry, anatomical connectivity, and requisite involvement in cocaine-memory reconsolidation. To test this hypothesis, male and female Sprague-Dawley rats received adeno-associated viruses to express Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) selectively in CRF neurons of the DR and injection cannulae directed at the BLA. The rats were trained to self-administer cocaine in a distinct environmental context then received extinction training in a different context. Next, they were briefly re-exposed to the cocaine-predictive context to destabilize (reactivate) cocaine memories. Intra-BLA infusions of the DREADD agonist deschloroclozapine (DCZ; 0.1 mM, 0.5 µL/hemisphere) immediately after memory reactivation attenuated cocaine-memory strength, relative to vehicle infusion. This was indicated by a selective, DCZ-induced and memory reactivation-dependent decrease in drug-seeking behavior in the cocaine-predictive context in DREADD-expressing males and females at test compared to respective controls. Notably, BLA-projecting DR CRF neurons that exhibited increased c-Fos expression during memory reconsolidation co-expressed the glutamatergic neuronal marker, vesicular glutamate transporter 3. Together, these findings suggest that the DRCRF → BLA circuit is engaged to maintain cocaine-memory strength after memory destabilization, and this phenomenon may be mediated by DR CRF and/or glutamate release in the BLA.

Anti-nociceptive effects of non-antibiotic derivatives of demeclocycline and doxycycline against formalin-induced pain stimulation

In previous studies, some tetracycline (TC) antibiotics showed potential as analgesic. We investigated here the analgesic activity of new non-antibiotic TC derivatives using the formalin-induced nociceptive pain model in adult C57BL/6 mice. Specifically, we tested the effects of i.p. injections of DDMC (5, 10, 20 mg.kg-1) and DDOX (10, 20, 40 mg.kg-1), which are non-antibiotic derivatives of demeclocycline and doxycycline, respectively. Repeated treatments with DDMC remarkably reduced nociceptive pain in both phases of the test, at 10 mg.kg-1 its efficacy was comparable to that of 10 mg.kg-1 of morphine. DDOX was also effective in this paradigm but intrinsically less potent than DDMC, exerting analgesic effects between 20 and 40 mg.kg-1. Interestingly, a single injection of DDMC (10 mg.kg-1) was sufficient to produce a robust anti-nociceptive effect similar to that of morphine. A single injection of DDOX (40 mg.kg-1) also produced anti-nociceptive effects but only in the second phase of the test. Noticeably, male mice exhibited a better analgesic response to DDMC (10 mg.kg-1) than females. A single injection of DDMC (10 mg.kg-1) and morphine but not of DDOX (40mg.kg-1), powerfully inhibited formalin-induced spinal cord c-Fos expression whereas both TC derivatives restrained the activation of Iba-1-immunoreactive cells, indicating a potential indirect effect on inflamed microglial cells. In summary, the non-antibiotic TCs, DDMC and DDOX, demonstrated notable analgesic efficacy against formalin-induced pain, suggesting their potential as alternatives for analgesic treatment.

Sesquiterpenoids from Inula britannica and their potential mechanism for immunomodulation

The immune system serves as a role of diseases, such as Parkinson's disease, and acute lung injury. An immunoregulatory activity-directed separation depended on phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Ion)-mediated Jurkat leukemic T cells was used for studying chemical constituents from Inula britannica L. in depth. Five previously undescribed aromatic sesquiterpenoid dimers inulabritanoids J−N (1−5) and a previously undescribed germacrane-type sesquiterpenoid britanicafanin F (6) were afforded from I. britannica as well as eight known sesquiterpenoids (7–14). Their structures were elucidated through 1D and 2D NMR, HRMS, and ECD spectra along with quantum chemical calculations. Immunomodulatory effects of compounds 1–14 were assayed in PMA plus Ion-mediated Jurkat cells, and indicated that compounds 8, 9, and 13 displayed significantly inhibitory effects toward IL-2 and IFN-γ. Further investigation of mechanism of action revealed that compound 13 inhibited phosphorylations of p38, ERK, and JNK to suppress c-Jun and c-Fos expressions, resulting in blocking the nuclear translocation of AP-1 (a complex of c-Jun and c-Fos) to regulate mRNA expressions of IL-2 and IFN-γ. Molecular docking analysis demonstrated that compound 13 could enter into the cavity of p38, ERK, and JNK, and from hydrogen bond interactions with Gly33, Lys53 Ser154, and Asp168 for p38, Lys54, Glu71, Ser153, and Asp167 for ERK, and Met149 and Asn152 for JNK, which supported the abovementioned results. These findings suggested that sesquiterpenoids from the genus Inula served as immunomodulators for treating diseases involved in immune and inflammatory responses.

Methylone regulates fear memory and amygdala activity: A potential treatment for posttraumatic stress disorder?

Methylone (3,4-methylenedioxy-N-methylcathinone) is a rapid-acting entactogen that has demonstrated significant benefits for patients with post-traumatic stress disorder (PTSD) and exhibits good tolerability in phase 1 clinical trials. Despite these promising results, its preclinical effects on fear memory regulation and the underlying mechanisms remain largely unexplored. This study aims to investigate the impact of methylone on auditory fear extinction and its influence on neuronal and synaptic activity in the basolateral amygdala (BLA). Using C57BL/6 mice, we employed an auditory fear conditioning paradigm along with immunofluorescent staining, extracellular electrophysiological recording, and chemogenetic techniques. The results revealed that administering methylone at a dosage of 10 mg/kg, in conjunction with extinction trials, significantly decreased the retrieval of both recent and remote fear memories. Additionally, methylone effectively inhibited the renewal of remote fear memories and blocked spontaneous recovery. It also reduced fear generalization to both context and tone. At the cellular level, methylone increased c-fos expression in the BLA and induced sustained elevations in long-term potentiation and long-term depression at the synaptic level. Furthermore, intra-BLA microinfusion of methylone directly enhanced the extinction memory. Chemogenetic activation of the BLA mimicked the effects of methylone, whereas chemogenetic inhibition blocked them. These findings suggest that methylone modulates fear memories through its action on the BLA. This preclinical study offers a knowledge base and critical insights into the potential future application of methylone for PTSD treatment.

Synapse-to-nucleus ERK-CREB transcriptional signaling requires dendrite-to-soma Ca2+propagation mediated by L-type voltage-gated Ca2+ channels.

The cAMP-response element binding protein (CREB) transcription factor controls the expression of the neuronal immediate early genes c-Fos, Arc, and Bdnf and is essential for long-lasting synaptic plasticity underlying learning and memory. Despite this critical role, there is still ongoing debate regarding the synaptic excitation-transcription (E-T) coupling mechanisms mediating CREB activation in the nucleus. Here we employed optical uncaging of glutamate to mimic synaptic excitation of distal dendrites in conjunction with simultaneous imaging of intracellular Ca2+ dynamics and transcriptional reporter gene expression to elucidate CREB E-T coupling mechanisms in hippocampal neurons cultured from both male and female rats. Using this approach, we found that CREB-dependent transcription was engaged following dendritic stimulation of N-methyl, D-aspartate receptors (NMDARs) only when Ca2+ signals propagated to the soma via subsequent activation of L-type voltage-gated Ca2+ channels resulting in activation of Extracellular signal-Regulated Kinase (ERK) MAP kinase signaling to sustain CREB phosphorylation in the nucleus. In contrast, dendrite-restricted Ca2+ signals generated by NMDARs failed to stimulate CREB-dependent transcription. Furthermore, Ca2+-CaM-dependent kinase (CaMK)-mediated signaling pathways that may transiently contribute to CREB-phosphorylation following stimulation were ultimately dispensable for downstream CREB-dependent transcription and c-Fos induction. These findings emphasize the essential role that L-type Ca2+ channels play in rapidly relaying signals over long distances from synapses located on distal dendrites to the nucleus to control gene expression.Significance Statement The transcription factor CREB controls gene expression programs required for long-lasting synaptic plasticity and learning and memory, yet the synapse-to-nucleus signaling mechanisms mediating CREB activation are still unclear. Using glutamate uncaging to mimic synaptic input to dendrites, this study shows that Ca2+ signals propagated to the soma by L-type voltage-gated Ca2+ channels engage the ERK MAP kinase cascade to mediate CREB phosphorylation and CREB-dependent transcription. In contrast, dendrite-restricted Ca2+ signals generated primarily by NMDARs failed to effectively engage this signaling pathway or CREB-dependent transcription. In addition, we found that while ERK and CaMK pathways may both contribute to increased CREB phosphorylation immediately following neuronal stimulation, sustained ERK signaling to CREB was necessary to effectively drive CREB-dependent transcription.

Nuciferine inhibits osteoclast formation through suppressing glycolysis metabolic programming and ROS production.

Nuciferine (NCF) is a bioactive compound from lotus leaves and has been proven to prevent osteoclastogenesis and ovariectomy-induced osteoporosis by our previous research. However, the underlying mechanism is still unclear. In this research, Raw264.7 cells were induced into osteoclast with or without NCF. CCK-8 and Edu assays were performed to detect the effects of 30 μM NCF on cell viability and proliferation. TRAP staining and bone resorption assays were performed to observe the role of NCF in osteoclastogenesis and bone resorption. RT-PCR and Western blot were performed to detect the effects of NCF on osteoclast-related genes, glycolysis-related genes, and reactive oxygen species (ROS)-related genes. Seahorse assays, lactate concentration and glucose consumption were performed to observe cell metabolism change. DCFH-DA fluorescent probe was used to detect ROS level. In this work, 30 μM NCF could not influence cell viability and cell proliferation. Osteoclast differentiation could be inhibited by 30 μM NCF. Bone resorption assay could also observe that bone resorption ability was successfully inhibited by 30 μM NCF. In seahorse assay, we discovered that NCF could decrease extracellular acid rate and increase oxygen consumption. RT-PCR and Western blot results showed that NCF could decrease the expression of hexokinase2, pyruvate kinase muscle 2, and lactate dehydrogenase A and that NCF could also weaken the concentration of lactate. However, pyruvate kinase muscle 2 activator (GC69716) and lactate addition could promote osteoclastogenesis and bone resorption and promote the expression of c-Fos and nuclear factor of activated T cells c1. Besides, NCF could also inhibit the production of ROS. In conclusion, NCF might inhibit osteoclast formation through inhibiting glycolysis metabolism and ROS production.

Hippocampal area CA2 activity supports social investigation following an acute social stress.

Neuronal activity in the hippocampus is critical for many types of memory acquisition and retrieval and influences an animal's response to stress. Moreover, the molecularly distinct principal neurons of hippocampal area CA2 are required for social recognition memory and aggression in mice. To interrogate the effects of stress on CA2-dependent behaviors, we chemogenetically manipulated neuronal activity in vivo during an acute, socially derived stressor and tested whether memory for the defeat was influenced. One day after an acute social defeat (aSD), defeated mice spent significantly less time investigating another mouse when compared to non-defeated control mice. We found that this avoidant phenotype persisted for up to one month following a single defeat encounter. When CA2 pyramidal neuron activity was inhibited with Gi-DREADD receptors during the defeat, subject mice exhibited a significantly higher amount of social avoidance one day later when compared to defeated littermates not expressing DREADDs. Moreover, CA2 inhibition during defeat caused a reduction in submissive defense behaviors in response to aggression. In vitro electrophysiology and tracing experiments revealed a circuit wherein CA2 neurons connect to caudal CA1 projection neurons that, in turn, project to corticolimbic regions including the anterior cingulate cortex. Finally, socially avoidant, defeated mice exhibited significant reductions in cFos expression in caudal hippocampal and limbic brain areas during a social investigation task 24 h after aSD. Taken together, these results indicate that CA2 neuronal activity is required to support behavioral resilience following an acute social stressor and that submissive defensive behavior during the defeat (vs. fleeing) is a predictor of future resilience to social stress. Furthermore, CA2 preferentially targets a population of caudal CA1 projection neurons that contact cortical brain regions where activity is modulated by an acute social stressor.

Mapping of c-Fos Expression in Rat Brain Sub/Regions Following Chronic Social Isolation: Effective Treatments of Olanzapine, Clozapine or Fluoxetine.

The c-Fos as a marker of cell activation is used to identify brain regions involved in stimuli processing. This review summarizes a pattern of c-Fos immunoreactivity and the overlapping brain sub/regions which may provide hints for the identification of neural circuits that underlie depressive- and anxiety-like behaviors of adult male rats following three and six weeks of chronic social isolation (CSIS), relative to controls, as well as the antipsychotic-like effects of olanzapine (Olz), and clozapine (Clz), and the antidepressant-like effect of fluoxetine (Flx) in CSIS relative to CSIS alone. Additionally, drug-treated controls relative to control rats were also characterized. The overlapping rat brain sub/regions with increased expression of c-Fos immunoreactivity following three or six weeks of CSIS were the retrosplenial granular cortex, c subregion, retrosplenial dysgranular cortex, dorsal dentate gyrus, paraventricular nucleus of the thalamus (posterior part, PVP), lateral/basolateral (LA/BL) complex of the amygdala, caudate putamen, and nucleus accumbens shell. Increased activity of the nucleus accumbens core following exposure of CSIS rats either to Olz, Clz, and Flx treatments was found, whereas these treatments in controls activated the LA/BL complex of the amygdala and PVP. We also outline sub/regions that might represent potential neuroanatomical targets for the aforementioned antipsychotics or antidepressant treatments.

CNSC-45. EXTRINSIC ELECTRICAL MODULATION IMPACTS GLIOBLASTOMA TUMOR GROWTH BY DISRUPTING ITS INTERCELLULAR COMMUNICATION

Abstract Glioblastoma (GBM) is an incurable primary malignant neoplasm of the central nervous system. GBM as an electrically active neoplasm diffusely colonizes healthy brain tissues, disrupting the neurotransmission balance and causing glioma-related epilepsy (GRE) in up to 50% of patients. Interestingly, GRE is an independent favorable prognostic factor for survival in GBM. The therapeutic induction of seizure may have a previously unexplored oncologic value in GBM. We have previously reported in CT-2A-bearing mice subjected to daily extrinsic electrical modulation (EEM) diminished tumor progression and increased overall survival relative to sham-treated mice, but the mechanism by which EEM produces its anti-tumor effect remains uncertain. Therefore, GBM-bearing mice (CT-2A cells xenografted into 5-7 weeks old C57BL/6 female mice, n=5/group) received either a single EEM of 2.0 millicoulombs (mC) or sham treatment. The mice were terminated at 3 hours, 24 hours, 4, 7, and 10 days post-EEM, and tumors and contralateral brain cortex were analyzed using bulk RNA-sequencing and Sequential immunofluorescence (SeqIFTM). SeqIFTM revealed how EEM activates neuronal activity via cFOS expression and induces tumor-specific cell damage through cleaved caspase-3 expression, without affecting healthy brain tissue. Furthermore, RNA-seq performed post-EEM revealed gene expression changes of not only synapse organization markers but also of several tumor-tumor and tumor-neuron connectivity key markers, such as growth-associated protein 43 (GAP43), neuroligin-3 (NLGN3), and glutamate receptors such as GRIA4 and GRIN1 between others. However, the expression of these markers diminished significantly 96h after-EEM, indicating a reduction in intercellular electrical communication by the inhibition of the neoplasm cells electrical integration with neuronal circuits by downregulating glutamatergic receptor expression, blocking the synapse-forming protein NLGN3, and the network-connecting protein GAP43. Taken together, our findings reveal how by the disruption of these critical interactions within the tumor microenvironment though EEM impacts on GBM growth making it more susceptible to therapeutic interventions.

CNSC-64. BEYOND THE LOCAL TUMOR MICROENVIRONMENT: CHARACTERIZING CHANGES TO NEURONAL ACTIVITY INDUCED BY PEDIATRIC HIGH-GRADE GLIOMA

Abstract Bidirectional electrochemical signaling between neurons and tumor cells is emerging as a critical regulator of pediatric high-grade glioma (HGG) pathology. Neuron-tumor interactions are known to induce local neuronal hyperactivity. However, recent work tracing the projections of these neuron-tumor networks revealed that HGG neuronal recruitment extends beyond the local tumor microenvironment (TME). These findings suggest that tumor-induced hyperexcitability may spread beyond the local TME and drive brain-wide circuit-level changes. To elucidate how non-tumor innervated brain regions are affected by TME hyperactivity we first characterized neuronal hyperactivity induced by a cortical patient-derived pediatric high-grade cell line in vitro on human iPSC-derived neurons. In vitro calcium imaging experiments demonstrated an increase in the number of calcium fluctuations in glioma co-cultured neurons. In parallel, multi-electrode recordings revealed increased population spiking activity and amplitude in neurons co-cultured with glioma cells. We then xenografted these HGG cells into the prefrontal cortex of mice and performed whole brain-clearing and cFos staining on tumor-bearing vs saline-injected control samples. As cFos is an immediate early gene activated by neuronal activity, we utilized the density of cFos expression as a readout-out of neuronal activation in our atlas-aligned segmented brain section analyses. We expectedly observed increased cFos expression within the ipsilateral primary and secondary motor region, which collectively comprise the local TME. Interestingly, we additionally observed elevated cFos in non-tumor bearing brain regions on both the ipsilateral and contralateral hemispheres, largely in layer 2/3 cortical neurons. These neurons form extensive cortico-cortical projections, and we coincidingly observed increased cFos expression in known cortical projection targets of the prefrontal xenograft location, such as somatosensory, visual, and retrosplenial cortices. Our findings suggest that tumor-induced neuronal hyperactivity extends beyond the local TME in a brain-wide manner and potentially follows established neuronal circuits, rendering these connected regions vulnerable to alterations in neuronal activity.

Linarin Identified as a Bioactive Compound of Lycii Cortex Ameliorates Insulin Resistance and Inflammation Through the c-FOS/ARG2 Signaling Axis.

Insulin resistance (IR) is a central pathophysiological process underlying numerous chronic metabolic disorders, including type 2 diabetes and obesity. Lycii Cortex, a widely used traditional Chinese herb, has demonstrated potential benefits in preventing and managing diabetes and IR. Whereas, the specific bioactive compounds responsible for these protective effects and their underlying mechanisms of action remain elusive. This study aimed to identify the bioactive components within Lycii Cortex that contribute to its anti-diabetic effects and to elucidate the molecular mechanisms underlying its beneficial actions on insulin resistance. Network pharmacology and molecular docking analyses were employed to identify the potential active compounds in Lycii Cortex and their corresponding target proteins. An invitro model of IR was established using palmitic acid (PA)-treated HepG2 cells. Cell viability was assessed using the CCK-8 assay, while glucose uptake was evaluated by 2-NBDG staining and extracellular glucose measurement. To validate the invitro findings, an invivo model of obesity-induced IR was established using high-fat diet (HFD)-fed mice. The network pharmacology analysis preliminarily identified 13 candidate chemicals and 10 hub LyC and IR-related genes (LIRRGs). Molecular docking analysis demonstrates that Linarin as the potential active component exhibits the greatest potential to target c-FOS for preventing obesity-induced IR. Enrichment analysis suggested that Linarin-targeted pathways are correlated with inflammation. Invitro experimental validation demonstrated that Linarin was capable of protecting against PA-induced IR in HepG2 cells evidenced by improving glucose uptake ability and reducing extracellular glucose content. Additionally, we found that Linarin ablated PA-induced increase in the expression of c-FOS and inflammatory cytokines. Furthermore, in PA-treated cells, silencing c-FOS markedly improved glucose consumption, and reduced inflammation and Arginase 2 (ARG2) expression. Similarly, as exposure to PA, silencing ARG2 also ameliorated glucose uptake and inflammation, while not affecting c-FOS expression. Invivo experiments further showed that Linarin administration remarkably improved glucose tolerance and insulin sensitivity, and reduced the fat mass and body weight in HFD-induced obese mice. In this study, Linarin has been identified as the bioactive compound of Lycii Cortex to ameliorate obesity-related IR and inflammation through the c-FOS/ARG2 signaling cascade. These findings underscore the therapeutic potential of Linarin and provide valuable insights into developing novel intervention strategies for type 2 diabetes therapy.

Nerve Growth Factor from Pancreatic Cancer Cells Promotes the Cancer Progression by Inducing Nerve Cell-Secreted Interleukin-6.

Pancreatic cancer (PC) is a cancer with a poor prognosis, and nerve growth factor (NGF) is involved in the pathogenesis of PC within the unknown exact role. Herein, SW1990 cells and PC12 cells were co-cultured using transwell co-culture system and subsequently revealed that NGF was overexpressed in SW1990 cells and promoted PC12 cell proliferation. Knockdown of NGF expression in SW1990 cells using lentiviral shRNA effectively inhibited NGF expression in SW1990 cells and reduced its stimulatory effect on PC12 cell proliferation. Additionally, NGF in SW1990 cells increased the expression of IL-6, dopamine, and c-FOS, as well as decreased the level of lactate dehydrogenase, in PC12 cells, whereas the inhibition of NGF expression significantly reduced the levels of IL-6, dopamine and c-FOS, indicating the critical role of IL-6/STAT3 signaling in PC progression. Finally, cell proliferation, migration, and invasion were assessed using cell counting kit-8, scratch, and Transwell assays, which showed that activated neurons promoted the proliferation, migration, invasion, and NGF secretion of SW1990 cells through the IL-6/STAT3 pathway. The results revealed that NGF secreted by PC cells played a pivotal role in PC progression via regulating activated neural cells-secreted IL-6, providing new theoretical insights for the treatment of PC.

Classification of psychedelics and psychoactive drugs based on brain-wide imaging of cellular c-Fos expression.

Psilocybin, ketamine, and MDMA are psychoactive compounds that exert behavioral effects with distinguishable but also overlapping features. The growing interest in using these compounds as therapeutics necessitates preclinical assays that can accurately screen psychedelics and related analogs. We posit that a promising approach may be to measure drug action on markers of neural plasticity in native brain tissues. We therefore developed a pipeline for drug classification using light sheet fluorescence microscopy of immediate early gene expression at cellular resolution followed by machine learning. We tested male and female mice with a panel of drugs, including psilocybin, ketamine, 5-MeO-DMT, 6-fluoro-DET, MDMA, acute fluoxetine, chronic fluoxetine, and vehicle. In one-versus-rest classification, the exact drug was identified with 67% accuracy, significantly above the chance level of 12.5%. In one-versus-one classifications, psilocybin was discriminated from 5-MeO-DMT, ketamine, MDMA, or acute fluoxetine with >95% accuracy. We used Shapley additive explanation to pinpoint the brain regions driving the machine learning predictions. Our results support a novel approach for characterizing and validating psychoactive drugs with psychedelic properties.

Persistent threat avoidance following negative reinforcement is not associated with elevated state anxiety.

Obsessive-Compulsive Disorder (OCD) is a debilitating illness consisting of obsessions and compulsions. OCD severity and treatment response are correlated with avoidant behaviors thought be performed to alleviate obsession-related anxiety. However, little is known about either the role of avoidance in the development of OCD or the interplay between anxiety states and avoidance behaviors. We have developed an instrumental negative reinforcement (i.e. active avoidance) paradigm in which mice must lever-press to avoid upcoming foot shocks. We show that mice (both sexes) can learn this task with high acquisition rates (75%) and that this behavior is largely stable when introducing uncertainty and modifying task structure. Furthermore, mice continue to perform avoidance responses on trials where lever pressing is not reinforced and increase response rates as they are maintained on this paradigm. With this paradigm, we did not find a relationship between negative reinforcement history and anxiety-related behaviors in well-established anxiety assays. Finally, we performed exploratory analyses to identify candidate regions involved in well-trained negative reinforcement using expression of the immediate early gene c-Fos. We detected correlated c-Fos expression in 1) cortico-striatal regions which regulate active avoidance in other paradigms and 2) amygdala circuits known to regulate conditioned defensive behaviors.Significance Statement Studies in patients with OCD suggest that compulsions are performed to avoid perceived threats and modulate anxiety tied to obsessions and/or compulsions. The negative reinforcement of avoidance and alleviated anxiety could therefore be a key driver of compulsive behaviors. However, there are still outstanding questions concerning the relationship between these two behaviors and the neural circuits involved in mediating negative reinforcement. We have developed an operant negative reinforcement paradigm in mice with discrete avoid and escape behaviors that can be learned without prior reward training with high throughput (75% acquisition) with responding that persists during nonreinforced trials. However, no differences were observed between negative reinforcement vs. unshocked and inescapably shocked controls, suggesting that continued negative reinforcement did not impact anxiety.

Learning-associated astrocyte ensembles regulate memory recall.

The physical manifestations of memory formation and recall are fundamental questions that remain unresolved1. At the cellular level, ensembles of neurons called engrams are activated by learning events and control memory recall1-5. Astrocytes are found in close proximity to neurons and engage in a range of activities that support neurotransmission and circuit plasticity6-10. Moreover, astrocytes exhibit experience-dependent plasticity11-13, although whether specific ensembles of astrocytes participate in memory recall remains obscure. Here we show that learning events induce c-Fos expression in a subset of hippocampal astrocytes, and that this subsequently regulates the function of the hippocampal circuitin mice. Intersectional labelling of astrocyte ensembles with c-Fos after learning events shows that they are closely affiliated with engram neurons, and reactivation of these astrocyte ensembles stimulates memory recall. At the molecular level, learning-associated astrocyte (LAA) ensembles exhibit elevated expression of nuclear factor I-A, and its selective deletion from this population suppresses memory recall. Taken together, our data identify LAA ensembles as a form of plasticity that is sufficient to provoke memory recall and indicate that astrocytes are an active component of the engram.

The Temporal Changes in Ankle Joint Pathology, Pain and Secondary Osteoporosis in Collagen-Induced Arthritis Rats.

Rheumatoid arthritis (RA) is a synovial inflammation-associated autoimmune disease with secondary osteoporosis. Pain is the most important symptom of RA, and some patients with well-controlled inflammation may still experience pain. To explore the relationship and dynamic changes between synovial inflammation and pain and bone destruction in collagen-induced arthritis (CIA) model rats, and to choose a better time window for drug treatment. The CIA model rats were constructed for 1, 2, 3, and 4-week groups. The changes were observed by joint swelling and behavioral assessment. The paw mechanical withdrawal threshold (PWT) was used for pain assessment. The micro-CT was used to assess joint injury and bone destruction. The biomechanics were performed to evaluate tension and compression test. The histological staining was used to observe ankle joint pathology. The immunohistochemical staining and Western blot were used to estimate the expression of calcitonin gene-related peptide (CGRP) and c-fos. The results showed that the degree of joint swelling, synovial hyperplasia, and inflammatory response were alleviated to varying extents over time. However, there were no significant changes in bone destruction, osteoclasts, or the maximum load of compression and tension. It showed secondary osteoporosis from the first week of the CIA model with no significant changes during the course of the experiment. There was no significant improvement in the PWT, and the expression of CGRP and c-fos was significantly increased over time, indicating hyperalgesia aggravation. Additionally, the result showed that repeated open-field tests might reduce the total distance of spontaneous movement. The results suggested that the pain and joint inflammation might not be synchronized, possibly related to post-inflammatory hyperalgesia. CIA model could be used for the study of pain, also relatively stable and suitable for the study of RA with secondary osteoporosis.

Resting-State Functional Magnetic Resonance Imaging Reveals the Effects of rTMS on Neural Activity and Brain Connectivity After Experimental Stroke.

Limited understanding of neurobiological mechanisms of repetitive transcranial magnetic stimulation (rTMS) prevents us from choosing optimal therapeutic regimen for patients to improve therapeutic efficiency. Resting-state functional magnetic resonance imaging (rs-fMRI) has been demonstrated to obtain comparable functional readouts across species. Intermittent and continuous theta burst stimulation were used to stimulate ipsilesional and contralesional hemisphere, respectively, during the subacute phase after stroke. We used a rat middle cerebral artery occlusion stroke model. The amplitude of low-frequency fluctuations and functional connectivity analyses of rs-fMRI were chosen to detect neuron activity and functional connectivity. The expression of neuron activation marker c-Fos and axonal plasticity marker GAP43 was examined by an immunochemistry method to corroborate the results of rs-fMRI. iTBS altered the long-term neuronal activity in bilateral sensorimotor cortex, whereas cTBS influenced immediate neuronal activity of bilateral sensorimotor cortex. In addition, cTBS enhanced interhemispheric and intrahemisheric functional connectivity in contralesional hemisphere, accompanied by axonal and dendritic remodeling in the perilesional cortical areas and contralesional homologous areas after large stroke. rTMS exerted complex effects on brain structural and functional connectivity in addition to affecting cortical excitability. cTBS promoted the compensatory effect of contralesional hemisphere after stroke with large lesions.

Expression of CCL2 signaling pathway genes in patients with periodontitis and atherosclerosis.

Periodontitis and atherosclerosis are chronic inflammatory diseases characterized by leukocyte infiltration. We investigated the expression of CCL4, CCR5, c-Jun, c-Fos, NF-κB, and CCL2 as well as the possible mechanism involved in the regulation of CCL2 in human periodontitis tissues and atherosclerotic aorta based on previous research on the CCL4/CCR5/c-Jun and c-Fos/CCL2 pathway leading to CCL2 expression in collagen-induced arthritis (CIA) rat. Sixty-five volunteers were recruited and the condition of their gingiva and coronary arteries were assessed. The subjects were divided into four groups: healthy control, chronic periodontitis (CP), coronary artery diseases (CAD), and noncoronary artery diseases (non-CAD). Total RNA was isolated from gingiva in periodontitis patients and control populations and from the aorta in patients with and without CAD. PCR was used to examine CCL4, CCR5, c-Jun, c-Fos, NF-κB, and CCL2 levels. The production of CCL2 in the gingiva and aorta was analyzed by immunostaining. PCR revealed that CCL4, CCR5, and CCL2 mRNA levels were increased in CP patients' gingivae and aortas from coronary artery bypass grafting (CABG) patients. Marked c-Jun, c-Fos, and NF-κB gene productions were detected in CP patients' gingivae but did not show statistical differences between the CAD and non-CAD groups. Stronger immunoreactivity against CCL2 was observed in periodontitis gingiva and aorta from CABG patients. Our findings suggest that the CCL4/CCR5/c-Jun and c-Fos/CCL2 pathways may be involved in CCL2 expression in periodontitis. CCL4, CCR5, and CCL2 might act as possible nodes to link the presence of periodontitis and atherosclerosis.