Mapping the neural circuit of a specific neuronal subclass is central to understanding the working mechanism of the brain. Currently, numerous types of transgenic mice expressing Cre recombinase have been engineered and...Mapping the neural circuit of a specific neuronal subclass is central to understanding the working mechanism of the brain. Currently, numerous types of transgenic mice expressing Cre recombinase have been engineered and widely used in neuroscience. To map the multilevel inputs into the neural circuit of a specific neuronal subpopulation, a Cre-dependent retrograde trans-multisynaptic tracer must be developed. The vaccine strain of Pseudorabies virus (PRV, Bartha strain) can infect neurons and spread in a retrograde manner in the neural circuit. In this study, we engineered the genome of PRV Bartha strain to prepare two new tracers, PRV676 and PRV829, by replacing the TK gene of PRV with the Cre-dependent expression cassette of the fluorescent protein gene and the TK gene. These two tracers can separately and Cre-dependently express EGFP and mRuby3 and produce progeny viruses in vitro and in vivo, which can help to map the multilevel inputs of a specific neuronal subpopulation expressing Cre. Collectively, our work provides two new tools for neuroscience research.
Selection of appropriate food is an ability that allows animals to make optimal foraging choices. However, the neural mechanisms that control this food selection remain unclear. The purpose of this study was to investiga...Selection of appropriate food is an ability that allows animals to make optimal foraging choices. However, the neural mechanisms that control this food selection remain unclear. The purpose of this study was to investigate the connection between memory and the feeding behavior of Drosophila melanogaster when two sucrose solutions with different concentrations are available. We placed flies into plates with 150 mM and 100 mM sucrose solutions and measured the preference for the 150 mM one. Flies preferred the 150 mM solution over the 100 mM when all 60 wells of the plate were filled with both solutions; this preference decreased when there were only 8 wells with food. Remarkably, prior exposure to a plate with all 60 wells filled with both solutions enhanced the preference for the 150 mM, even when there were only 8 wells with food. We found that the memory-related gene rut and the dopamine D1 receptor on the mushroom body were required to enhance the preference after the prior exposure. These findings show that memory acquired through experiencing both solutions is stored in the mushroom body optimizing the food selection process.
Elevated plasma homocysteine (Hcy) levels lead to hyperhomocysteinemia, a condition associated with various neurological disorders affecting multiple brain regions, including the hippocampus. In this study, we investigat...Elevated plasma homocysteine (Hcy) levels lead to hyperhomocysteinemia, a condition associated with various neurological disorders affecting multiple brain regions, including the hippocampus. In this study, we investigated the effects of exposing cultured rat hippocampal neurons to Hcy concentrations corresponding to mild, moderate, and severe hyperhomocysteinemia. A short 24-hour exposure had minimal effects, whereas prolonged exposure up to 14 days moderately enhanced hippocampal excitability without altering the gene expression of voltage-dependent calcium, sodium, or potassium channels or intracellular calcium levels. These findings suggest that Hcy-induced changes in neuronal excitability may contribute to neuropathologies associated with hyperhomocysteinemia.
Fear extinction training in rodents decreases fear responses, providing a model for the development of post-traumatic stress disorder therapeutics. Fear memory recall reactivates the consolidated fear memory trace across...Fear extinction training in rodents decreases fear responses, providing a model for the development of post-traumatic stress disorder therapeutics. Fear memory recall reactivates the consolidated fear memory trace across multiple brain regions, and several studies have suggested that these recall-activated neurons are re-engaged during extinction. However, the molecular mechanisms linking this reactivation to extinction remain largely elusive. Here, we investigated the role of N-Methyl-D-Aspartate receptors (NMDARs) in remote memory recall-activated neurons within the basolateral amygdala and the medial prefrontal cortex during extinction training in mice. We found that Grin1 knockdown in these specific ensembles impaired extinction of remote fear memory, but did not reduce their reactivation during retrieval of the extinguished memory. These data suggest that while reactivation of these neuronal populations persists, their NMDARs are crucial for driving the synaptic plasticity needed to extinguish remote fear memories.
BACKGROUND: Parkinson's disease (PD), a globally prevalent neurodegenerative disorder, has been implicated with oxidative stress (OS) as a central pathomechanism. Excessive reactive oxygen species (ROS) trigger neuronal...BACKGROUND: Parkinson's disease (PD), a globally prevalent neurodegenerative disorder, has been implicated with oxidative stress (OS) as a central pathomechanism. Excessive reactive oxygen species (ROS) trigger neuronal damage and may induce disulfidptosis-a novel cell death modality not yet characterized in PD pathogenesis. METHOD: Integrated bioinformatics analyses were conducted using GEO datasets to identify PD-associated differentially expressed genes (DEGs). These datasets were subjected to: immune infiltration analysis, gene set enrichment analysis (GSEA), weighted gene co-expression network analysis (WGCNA), intersection analysis of oxidative stress-related genes (ORGs) and disulfidptosis-related genes (DRGs) for functional enrichment annotation. Following hub gene identification, diagnostic performance was validated using independent cohorts. LASSO regression was applied for feature selection, with subsequent experimental validation in MPTP-induced PD mouse models. Single-cell transcriptomic profiling and molecular docking studies were performed to map target gene expression and assess drug-target interactions. RESULT: A total of 1615 PD DEGs and 200 WGCNA DEGs were obtained, and the intersection with ORGs and DRGs resulted in 202 DEORGs, 11 DEDRGs, and 5 DED-ORGs (NDUFS2, LRPPRC, NDUFS1, GLUD1, and MYH6). These genes are mainly associated with oxidative stress, the respiratory electron transport chain, the ATP metabolic process, oxidative phosphorylation, mitochondrial respiration, and the TCA cycle. 10 hub genes have good diagnostic value, including in the validation dataset (AUC ≥ 0.507). LASSO analysis of hub genes yielded a total of 6 target genes, ACO2, CYCS, HSPA9, SNCA, SDHA, and VDAC1. In the MPTP-induced PD mice model, the expression of ACO2, HSPA9, and SDHA was decreased while the expression of CYCS, SNCA, and VDAC1 was increased, and the expression of the 5 DED-ORGs was decreased. Additionally, it was discovered that N-Acetylcysteine (NAC) could inhibit the occurrence of disulfidptosis in the MPTP-induced PD model. Subsequently, the distribution of target genes with AUC > 0.7 in different cell types of the brain was analyzed. Finally, molecular docking was performed between the anti-PD drugs entering clinical phase IV and the target genes. LRPPRC has low binding energy and strong affinity with duloxetine and donepezil, with binding energies of -7.6 kcal/mol and - 8.7 kcal/mol, respectively. CONCLUSION: This study elucidates the pathogenic role of OS-induced disulfidptosis in PD progression. By identifying novel diagnostic biomarkers (e.g., DED-ORGs) and therapeutic targets (e.g., LRPPRC), our findings provide a mechanistic framework for PD management and lay the groundwork for future therapeutic development.
In mammalian brains, astroglia presence near glutamatergic excitatory synapses has generated the term "tripartite" junctions, based on the close association of astrocytic processes near the active zone formed by presynap...In mammalian brains, astroglia presence near glutamatergic excitatory synapses has generated the term "tripartite" junctions, based on the close association of astrocytic processes near the active zone formed by presynaptic axonal terminal and postsynaptic dendritic spines. One major function of these astrocytic processes is to take up glutamate that spill out of the synaptic cleft during activity, via glutamate transporters located on astroglial plasma membrane. Comapred to other regions of the brain, the cerebellar Purkinje spines in the molecular layer are virtually completely ensheathed by Bergman glia, a special type of astrocyte, unique to cerebellum. The present electron microscopy study classifies these peri-synaptic astrocytic processes (PAP) ensheathing the Purkinje spine synapses into three types based on structural criteria: (1) Type 1- astrocytic process is situated at the edge of the synaptic cleft immediately next to the synaptic active zone. Under fast perfusion fixation conditions where synapses were under resting states, ~ 58% of the PAP's were scored as Type 1. The occurrence frequency of Type 1 PAP significantly decreased to 25% upon a 5-8 min delay in perfusion fixation, where synapses were under stimulated states. (2) Type 2- astrocytic process covers part of the postsynaptic membrane containing the postsynaptic density (PSD), so that this part of the PSD is separated from its presynaptic terminal. Occurrence frequency of Type 2 PAP's significantly increased from ~ 14% under fast perfusion fixation to 31% upon delayed perfusion fixation, and the average length of the PSD edge covered by astroglia increased from 41 nm to 57 nm upon delayed perfusion fixation. (3) Type 3- astrocytic process is situated some distance away from the active zone, while the presynaptic axon terminal extends to enwrap the spine beyond the active zone. Occurrence frequency of Type 3 PAP's increased from 28 to 43% upon delayed perfusion fixation, and the average length between apposed axon terminal and spine beyond the synaptic cleft significantly increased from 98 to 209 nm upon delayed perfusion fixation. Thus, upon stimulation, the tripartite synaptic junctions undergo dynamic structural changes with the astrocytic processes moving into the open cleft to cover the exposed postsynaptic membrane containing PSD, the presynaptic axon terminals extending to wrap the postsynaptic spine beyond the synaptic cleft. Both structural changes may facilitate glutamate uptake to clear the transmitter spilled out from the synaptic cleft during intense activity and prevent damage from overstimulation.
Klotho, a well-known aging suppressor protein, has been implicated in neuroprotection and the regulation of neuronal senescence. While previous studies have demonstrated its anti-aging properties in human brain organoids...Klotho, a well-known aging suppressor protein, has been implicated in neuroprotection and the regulation of neuronal senescence. While previous studies have demonstrated its anti-aging properties in human brain organoids, its potential to mitigate neurodegenerative processes triggered by β-amyloid remains underexplored. In this study, we utilised human induced pluripotent stem cells (iPSCs) engineered with a doxycycline-inducible system to overexpress KLOTHO and generated 2D cortical neuron cultures from these cells. These neurons were next exposed to pre-aggregated β-amyloid 1-42 oligomers to model the neurotoxicity associated with Alzheimer's disease. Our data reveal that upregulation of KLOTHO significantly reduced β-amyloid-induced neuronal degeneration and apoptosis, as evidenced by decreased cleaved caspase-3 expression and preservation of axonal integrity. Additionally, KLOTHO overexpression prevented the loss of dendritic branching and mitigated reductions in axonal diameter, hallmark features of neurodegenerative pathology. These results highlight Klotho's protective role against β-amyloid-induced neurotoxicity in human cortical neurons and suggest that its age-related decline may contribute to neurodegenerative diseases such as Alzheimer's disease. Our findings underscore the therapeutic potential of Klotho-based interventions in mitigating age-associated neurodegenerative processes.
Mutations in CACNA1C, the gene encoding Ca1.2 voltage-gated calcium channels, are associated with a spectrum of disorders, including Timothy syndrome and other neurodevelopmental and cardiac conditions. In this study, we...Mutations in CACNA1C, the gene encoding Ca1.2 voltage-gated calcium channels, are associated with a spectrum of disorders, including Timothy syndrome and other neurodevelopmental and cardiac conditions. In this study, we report a child with a de novo heterozygous missense variant (c.1973T > C; L658P) in CACNA1C, presenting with refractory epilepsy, global developmental delay, hypotonia, and multiple systemic abnormalities, but without overt cardiac dysfunction. Electrophysiological analysis of the recombinant Ca1.2 L658P variant revealed profound gating alterations, most notably a significant hyperpolarizing shift in the voltage dependence of activation and inactivation. Additionally, molecular modeling suggested that the L658P mutation disrupts interactions within the IIS5 transmembrane segment, reducing the energy barrier for state transitions and facilitating channel opening at more negative voltages. These findings establish L658P as a pathogenic CACNA1C variant primarily associated with severe neurological dysfunction and expands the phenotypic spectrum of CACNA1C-related disorders.
Adult hippocampal neurogenesis is inhibited by chronic psychological stress and impaired neurogenesis underlies stress-related psychological disorders. We previously reported that chronic restraint stress (CRS) evokes au...Adult hippocampal neurogenesis is inhibited by chronic psychological stress and impaired neurogenesis underlies stress-related psychological disorders. We previously reported that chronic restraint stress (CRS) evokes autophagic death of adult hippocampal neural stem cells (NSCs) while NSC-specific deletion of Atg7 prevents death of NSCs. Examination of cognitive ability and mood regulation next day of the termination of stress showed normal hippocampal function in mice deficient of Atg7. However, it was not investigated whether the preservation of NSC pool alleviates hippocampal neuronal alterations. Here, we show that CRS increased c-Fos-positive, activated neurons in the granule cell layer and decreased spine density of CA3 neurons in the hippocampus, and these hippocampal neuronal deficits were prevented by NSC-specific deletion of Atg7. Of note, our observation was conducted right after the termination of CRS. Therefore, our results suggest that the detrimental effects of stress on hippocampal neurons can be buffered by NSCs independent of neurogenesis and NSCs are essential to the hippocampal function both through the neurogenesis-dependent developmental process and by direct regulation of neural activation.
Alpha-bisabolol and camphene have demonstrated analgesic effects in inflammatory pain models by blocking Cav3.2 calcium channels. As the pain pathway overlaps with mechanisms for itch, and because Cav3.2 channels have be...Alpha-bisabolol and camphene have demonstrated analgesic effects in inflammatory pain models by blocking Cav3.2 calcium channels. As the pain pathway overlaps with mechanisms for itch, and because Cav3.2 channels have been associated with itch in our previous work, we aimed to investigate the potential anti-itch effects of these two terpenes. Although both terpenes failed to show anti-pruritogenic properties when dissolved in aqueous PBS, when diluted in Hydroxypropyl-beta-cyclodextrin their bioactivity significantly increased. Both compounds significantly reduced scratching in the histaminergic itch model, whether administered subcutaneously or intraperitoneally. Camphene reduced itching in the non-histaminergic model regardless of the route of administration, whereas alpha-bisabolol did not alleviate chloroquine-induced itching. When tested in Cav3.2-/- mice, neither camphene nor alpha-bisabolol significantly reduced histamine-induced scratching behavior. This suggests that the anti-pruritic actions of these terpenes may involve Cav3.2 block to mitigate itch.
Psilocybin, a psychedelic compound found in specific hallucinogenic mushrooms, is known to induce changes in visual perception and experience in humans. However, there is little knowledge of the molecular mechanisms thro...Psilocybin, a psychedelic compound found in specific hallucinogenic mushrooms, is known to induce changes in visual perception and experience in humans. However, there is little knowledge of the molecular mechanisms through which psilocybin affects vision-associated regions in the brain, such as the visual cortex. The current study determined both psilocybin-induced and experience-dependent changes (exposure to light) in visual cortex gene expression in mice. Of great interest, psilocybin induced robust gene expression changes in the visual cortex that closely mirror light-induced gene expression changes, even when the mice are kept in the dark. These gene expression changes correspond to specific molecular pathways, including synaptic functioning, and represent genes expressed in specific subtypes of neurons. In addition, exposure to both psilocybin and light induced synergetic changes in genes involved in epigenetic programming. Overall, the study determined that psilocybin induces robust changes in gene expression in the visual cortex that may have functional consequences in visual perception both in the absence and in synergy with visual experience.
To investigate the effects of moderate ethanol exposure on glucose metabolism in APP/PS1 mice, an early-onset Alzheimer's disease (AD) mouse model, we employed an fluoro-deoxy-glucose (FDG)-micro-positron emission tomogr...To investigate the effects of moderate ethanol exposure on glucose metabolism in APP/PS1 mice, an early-onset Alzheimer's disease (AD) mouse model, we employed an fluoro-deoxy-glucose (FDG)-micro-positron emission tomography (PET). We also utilized the comprehensive lab animal monitoring system (CLAMS) to measure whole-body energy expenditure and respiratory exchange ratio (RER). We found that ethanol exposure increased glucose metabolism in the brain as measured by FDG-PET. Also, CLAMS data indicated a decrease in RER, suggesting a shift toward fat utilization as the primary energy source. Following ethanol exposure in APP/PS1 mice, these findings reveal a distinct metabolic difference between brain and peripheral tissues.
Peptide drug technologies offer powerful approaches to develop potent and selective lead molecules for therapeutic and research applications. However, new and optimized delivery approaches are necessary to overcome curre...Peptide drug technologies offer powerful approaches to develop potent and selective lead molecules for therapeutic and research applications. However, new and optimized delivery approaches are necessary to overcome current pitfalls including fast degradation in cells and tissue. Extracellular signal-regulated kinases 1/2 (ERK1/2) exemplifies proteins that play crucial and varied roles within distinct cellular compartments. Here, we established an innovative method, based on viral vectors, which utilizes the endogenous biogenesis of neurotrophins to deliver and express a biologically active peptide to attenuate specifically ERK1/2 nuclear functions in specific brain area of the adult forebrain. In contrast to our hypothesis, nuclear functions of ERK1/2 in the forebrain are fundamental for the extinction of associative-aversive memories, but not for acquisition, nor for retrieval of these memories. Our research demonstrates the feasibility and applicability of viral vectors to deliver a peptide of interest to manipulate specific molecular processes and/or protein interactions in specific tissue.
Neurexins are presynaptic plasma membrane proteins that regulate key aspects of synapse physiology through the formation of transcellular complexes with postsynaptic ligands, including neuroligins (Nlgns). Each neurexin...Neurexins are presynaptic plasma membrane proteins that regulate key aspects of synapse physiology through the formation of transcellular complexes with postsynaptic ligands, including neuroligins (Nlgns). Each neurexin gene (NRXN1-3) generates two main alternative-spliced transcripts that generate alpha and beta-Nrxn isoforms differing in their extracellular domains. Mutations in NRXN1 are associated with autism and other neurodevelopmental disorders. However, whether dysfunction of NRXN1 occurs through common or isoform-specific postsynaptic partners for alpha- and beta-Nrxn1 is not completely known. The association of Nrxn1 proteins with postsynaptic partners has been mostly analysed in experiments that test binding, but Nrxn proteins must interact with Nlgns in opposing cells, which requires transcellular oligomerization. Here, we studied the interactions of Nrxn1/Nlgn pairs across the synapse and identified the type of association affected in a mouse model of autism. We found that beta-Nrxn1 can be recruited at synaptic contacts by glutamatergic Nlgn1 and GABAergic Nlgn2, whereas alpha-Nrxn1 is a presynaptic partner of Nlgn2. Insertion of alternative spliced segment 4 (AS4) negatively modulates the presynaptic recruitment of Nrxn1 by Nlgns. These data obtained in transcellular assays help clarify previous knowledge based on the ability of Nrxn1 to bind to Nlgns. Interestingly, we found that a mutant beta-Nrxn1 shows ligand restriction for glutamatergic Nlgn1 in the brain of a mouse model of autism. These findings suggest that autism-associated mutations affecting beta-Nrxn1 can act through specific synaptic partners that may be different from those of its alpha-Nrxn1 counterparts.
Animals adaptively regulate aversive memories in safe environments through extinction, a process central to exposure therapy for anxiety disorders. The limbic thalamus controls cognitive function in concert with intercon...Animals adaptively regulate aversive memories in safe environments through extinction, a process central to exposure therapy for anxiety disorders. The limbic thalamus controls cognitive function in concert with interconnected cortical and limbic structures. Though medial prefrontal (mPFC) afferents to the limbic thalamus regulate aversive memory, the functional role of limbic thalamus efferents to mPFC is unclear. Here, we investigated the roles of thalamic nuclei, the reuniens (RE) and mediodorsal (MD) thalamus, projecting to the medial prefrontal cortex (mPFC) in aversive memory conditioning and extinction in male mice. Using retrograde tracing, we demonstrated that ventromedial PFC (vmPFC)- and dorsomedial PFC (dmPFC)-projecting neurons are topologically segregated within the RE and MD. Fiber photometry revealed that both RE→vmPFC and MD→vmPFC neurons respond to aversive stimuli. Notably, RE→vmPFC neurons develop shock-associated cue (CS+) response during aversive conditioning. During extinction, RE→vmPFC neurons exhibited a biphasic response to CS+, while MD→vmPFC neurons showed no cue-evoked activity. Neither optogenetic activation nor inactivation of these populations altered freezing behavior during extinction compared to controls. Collectively, these findings indicate that RE→vmPFC neurons encode aversive cue information during extinction but are dispensable for behavioral modulation. This study highlights the distinct contributions of limbic thalamus-PFC circuits to aversive memory processing.
Following intracerebral hemorrhage, mitigating oxidative stress and removing excess iron are critical strategies for reducing secondary brain injury and improving neurological outcomes. In vitro, we synthesized quercetin...Following intracerebral hemorrhage, mitigating oxidative stress and removing excess iron are critical strategies for reducing secondary brain injury and improving neurological outcomes. In vitro, we synthesized quercetin-ethylenediamine carbon quantum dots (QECQDs) with diameters of 2-11 nm and found that QECQDs effectively scavenge ABTS+· and DPPH· free radicals, defending HT22 cells against hemin-induced oxidative stress. In vivo, QECQDs predominantly accumulate in the pia mater, subarachnoid space, and dura mater after intrathecal injection. Compared to the ICH injury group, QECQDs treatment effectively improves cerebral blood flow, inhibits oxidative stress damage, and reduces neuron death. Importantly, QECQDs treatment reduced hemorrhage volume, alleviated edema, and improved neurological function. This lays a foundation for developing multi-target drugs for treating ICH.
Transient receptor potential melastatin type 2 (TRPM2) is a nonselective cation channel involved in synaptic plasticity. We investigated its role in contextual fear conditioning and extinction of conditioned fear using T...Transient receptor potential melastatin type 2 (TRPM2) is a nonselective cation channel involved in synaptic plasticity. We investigated its role in contextual fear conditioning and extinction of conditioned fear using Trpm2-deficient (Trpm2) mice. Trpm2 mice exhibited reduced acquisition of contextual fear memory during conditioning but had an intact freezing response to conditioning context 24 h after conditioning. They also showed a reduced freezing response to extinction training, indicating facilitated extinction. Consistent with this, infusion of flufenamic acid (FFA), a TRPM2 antagonist, into the dentate gyrus (DG) of the hippocampus in fear-conditioned mice facilitated extinction of contextual fear. The enhanced extinction in Trpm2 and FFA-treated mice was associated with down-regulation of immediate-early genes (IEGs) including Npas4, c-Fos, Arc and Egr1 in the hippocampus after extinction training. Our results indicate that TRPM2 plays a positive role in retention of contextual fear memory by modulating neuronal activity in the hippocampus, and suggest that TRPM2 activity could potentially be targeted to strengthen extinction-based exposure therapies for post-traumatic stress disorder (PTSD).
The TREM1 receptor, a member of the TREMs family, is expressed by myeloid cells and functions as an initiator or enhancer of the inflammatory response, playing a pivotal role in the regulation of inflammation. In recent...The TREM1 receptor, a member of the TREMs family, is expressed by myeloid cells and functions as an initiator or enhancer of the inflammatory response, playing a pivotal role in the regulation of inflammation. In recent years, it has been found that TREM1-mediated inflammatory response is involved in the regulation of pain-related diseases. This article provides an extensive review on the structural characteristics and distribution patterns, ligand, signaling pathways, inhibitors, and pathophysiological roles of TREM1 in pain disorders aiming to further elucidate its biological function and offer novel insights for clinical interventions targeting pain-related diseases.