Dendritic Spines Research Articles

Age-related loss of large dendritic spines in the precuneus is statistically mediated by proteins which are predicted targets of existing drugs.

Preservation of dendritic spines is a putative mechanism of protection against cognitive impairment despite development of Alzheimer Disease (AD)-related pathologies. Aging, the chief late-onset AD risk factor, is associated with dendritic spine loss in select brain areas. However, no study to our knowledge has observed this effect in precuneus, an area selectively vulnerable to early accumulation of AD-related pathology. We therefore quantified dendritic spine density in precuneus from 98 subjects without evidence of neurocognitive decline, spanning ages 20-96, and found a significant negative correlation between age and large dendritic spine density. In these same subjects, we conducted liquid chromatography-tandem mass spectrometry of >5000 proteins and identified 203 proteins which statistically mediate the effect of age on large dendritic spine density. Using computational pharmacology, we identified ten drugs which are predicted to target these mediators, informing future studies designed to test their effects on age-related dendritic spine loss and cognitive decline.

Synapses and dendritic spines are eliminated in the primary visual cortex of mice subjected to chronic intraocular pressure elevation

Synaptic plasticity is essential for maintaining neuronal function in the central nervous system and serves as a critical indicator of the effects of neurodegenerative disease. Glaucoma directly impairs retinal ganglion cells and their axons, leading to axonal transport dysfuntion, subsequently causing secondary damage to anterior or posterior ends of the visual system. Accordingly, recent evidence indicates that glaucoma is a degenerative disease of the central nervous system that causes damage throughout the visual pathway. However, the effects of glaucoma on synaptic plasticity in the primary visual cortex remain unclear. In this study, we established a mouse model of unilateral chronic ocular hypertension by injecting magnetic microbeads into the anterior chamber of one eye. We found that, after 4 weeks of chronic ocular hypertension, the neuronal somas were smaller in the superior colliculus and lateral geniculate body regions of the brain contralateral to the affected eye. This was accompanied by glial cell activation and increased expression of inflammatory factors. After 8 weeks of ocular hypertension, we observed a reduction in the number of excitatory and inhibitory synapses, dendritic spines, and activation of glial cells in the primary visual cortex contralateral to the affected eye. These findings suggest that glaucoma not only directly damages the retina but also induces alterations in synapses and dendritic spines in the primary visual cortex, providing new insights into the pathogenesis of glaucoma.

Contextual Fear Conditioning Uncovers Spine and Learning Deficits in the Retrosplenial Cortex in a Familial Alzheimer’s Disease Mouse Model

While excitatory synapse loss is documented in the brains of Alzheimer’s Disease (AD) patients, its role in episodic learning decline, one of the first evident AD symptoms, is unclear. The retrosplenial cortex (RSC), a cortical structure required for episodic learning, provides an ideal entry point to study the role of synapse loss in cognitive decline in AD since it is a site of high amyloid load and dysfunctional early in AD progression. Further, excitatory synapse assembly, both turnover and clustered synapse formation, is highly correlated with episodic learning performance in the RSC. We hypothesize alterations in synapse assembly in the RSC of AD patients contribute to early cognitive decline in the disease. To address this hypothesis, we examined both excitatory synapse density in the RSC and contextual learning in a familial AD (fAD) mouse model, an early onset amyloid model of AD. Importantly, we find age-dependent excitatory synapse loss in the RSC of fAD mice, as measured by imaging and quantification of GFP-labeled dendritic spines. Further, prior to frank spine loss in 5-month-old fAD mice we found episodic learning deficits, as measured by contextual fear conditioning (CFC). Using this early time point, we examined spine dynamics in vivo with MP imaging in the RSC of fAD mice or controls while engaged in CFC learning. Interestingly, we find deficits both in spine turnover and clustered spine formation and a loss of correlations of these spine metrics with CFC performance, suggesting aberrant spine dynamics may be causal in episodic learning deficits in this AD model, consistent with our hypothesis. Future studies will pharmacologically target these aberrant spine dynamics in fAD mice in an effort to strengthen the causal link between alterations in synapse assembly and cognitive decline in AD as well as provide potential therapeutic approaches to reverse early stages of the disease.

Development and evaluation of an autism pig model.

Developing cost-effective and disease-relevant animal models is essential for advancing biomedical research into human disorders. Here we investigate the feasibility of a pig model for autism spectrum disorder (ASD) using embryonic exposure to valproic acid (VPA), an antiepileptic drug known to increase ASD risk. We established experimental paradigms to assess the behavioral characteristics of these pig models. Administration of VPA to Bama miniature pigs (Sus scrofa domestica) during critical embryonic stages resulted in abnormal gait, increased anxiety levels, reduced learning capabilities and altered social patterns, while largely preserving social preference of treated piglets. Notably, we detected significant neuroanatomical changes in cortical regions associated with ASD in the VPA-treated pigs, including cortical malformation, increased neuronal soma size, decreased dendritic complexity and reduced dendritic spine maturation. Transcriptome analysis of the prefrontal cortex of VPA-treated pigs further revealed substantial alterations in the expression of genes linked to ASD, especially genes of the dopamine signaling pathway, highlighting the model's relevance and potential for shedding light on ASD's underlying neuropathological and molecular mechanisms. These findings suggest that pig models could serve as a promising alternative to traditional rodent models and provide a more ethical substitute for the use of primates in translational research on neurodevelopmental disorders.

RBIO-09. EFFECTS OF RADIATION ON THE HIPPOCAMPUS AND HIPPOCAMPAL NEUROGENESIS: A SYSTEMATIC REVIEW OF INJURY MECHANISMS AND INTERVENTION STRATEGIES

Abstract PURPOSE To understand how radiation affects the hippocampus and hippocampal neurogenesis. Radiotherapy is a common modality in the treatment of brain tumors that targets malignant cells with radiation. However, unintended iatrogenic injury may extend to adjacent healthy cells. The hippocampus is a particular area of concern, given its vital role in memory and learning and capacity for neurogenesis. METHODS We searched the PubMed, Embase, and Web of Science databases for studies assessing the effects of radiation on the hippocampus and hippocampal neurogenesis, adhering to PRISMA guidelines. Animal studies were evaluated according to the ARRIVE guidelines for pre-clinical studies. The SYRCLE risk of bias tool was utilized to examine potential bias among studies. Study characteristics, including study design, sample size, experimental methods, radiation dose, and results were collected and synthesized. RESULTS Ninety-two studies met inclusion criteria, and the majority of studies investigated rat and/or mouse models (n = 91, 98.9%). Across studies, cranial irradiation was consistently found to be associated with detrimental effects on hippocampal function and structure through molecular alterations to gene and protein expression, neuronal cell apoptosis, disruption of dendritic spines, and initiation of pro-inflammatory processes. The consequences for neurogenesis were mostly focused on the dentate gyrus of the hippocampus. These effects manifested as cognitive deficits in hippocampus-dependent tasks such as memory and learning. Behavioral (e.g., running) and pharmacological (e.g., PPARα agonists) interventions against the deleterious effects of radiation suggest the potential to address radiation-induced hippocampal disruption and cognitive deficits. The majority of studies were found to have an unclear risk of bias. CONCLUSIONS This systematic review highlights the effects of cranial radiation on hippocampal neurogenesis and their association with disruptions in memory and learning. Future research should continue to focus on delivering radiotherapy that spares susceptible regions in the brain, preserving patients’ cognitive function and quality of life.

LPM682000012, a Synthetic Neuroactive Steroid That Ameliorates Epileptic Seizures by Downregulating the Serpina3n/NF-κB Signaling Pathway

Epilepsy is characterized by abnormal neuronal firing in the brain. Several therapeutic strategies exist for epilepsy; however, several patients remain poorly treated. Therefore, the development of effective treatments remains a high priority in the field. Neuroactive steroids can potentiate extra-synaptic and synaptic GABAA receptors, thereby providing therapeutic benefits relative to benzodiazepines. This research study investigated the therapeutic effectiveness and underlying mechanisms of LPM682000012, a new synthetic neuroactive steroid-positive allosteric modulator (PAM) of GABAA receptors employed for treating epilepsy. Acute and chronic rat epilepsy models were established to identify the anti-seizure potency of LPM682000012. The dose-dependent sedative effects of LPM682000012 and Ganaxolone in normal rats were evaluated, which revealed that they both dose-dependently alleviated acute epileptic seizure in the pentylenetetrazol (PTZ)-mediated seizure model. Furthermore, LPM682000012 indicated an enhanced safety profile than Ganaxolone. Moreover, LPM682000012 also indicated therapeutic effects in the kainic acid (KA)-induced chronic spontaneous seizure model. Morphologically, LPM682000012 decreased neuronal loss in the hippocampal CA1 and CA3 regions and increased dendritic spine density in the CA1 region. In addition, mechanical analyses, including transcriptomics, Western blot, and proteomics analyses, revealed that the Serpina3n/NF-κB signaling pathway was up-regulated in epileptic rat hippocampal tissue, and LPM682000012 treatment reversed these changes. In summary, this report demonstrated that the novel neurosteroid GABAA PAM LPM682000012 activated the synaptic and extra-synaptic GABAA receptors and alleviated KA-induced neuronal loss and synaptic remodeling, potentially by down-regulating the Serpina3n/NF-κB signaling pathways. The results provide evidence that LPM682000012 is a potential anti-seizure pharmacotherapy candidate for epilepsy and warrants further research.

PKA regulation of neuronal function requires the dissociation of catalytic subunits from regulatory subunits

Protein kinase A (PKA) plays essential roles in diverse cellular functions. However, the spatiotemporal dynamics of endogenous PKA upon activation remain debated. The classical model predicts that PKA catalytic subunits dissociate from regulatory subunits in the presence of cAMP, whereas a second model proposes that catalytic subunits remain associated with regulatory subunits following physiological activation. Here, we report that different PKA subtypes, as defined by the regulatory subunit, exhibit distinct subcellular localization at rest in CA1 neurons of cultured hippocampal slices. Nevertheless, when all tested PKA subtypes are activated by norepinephrine, presumably via the β-adrenergic receptor, catalytic subunits translocate to dendritic spines but regulatory subunits remain unmoved. These differential spatial dynamics between the subunits indicate that at least a significant fraction of PKA dissociates. Furthermore, PKA-dependent regulation of synaptic plasticity and transmission can be supported only by wildtype, dissociable PKA, but not by inseparable PKA. These results indicate that endogenous PKA regulatory and catalytic subunits dissociate to achieve PKA function in neurons.

PKA regulation of neuronal function requires the dissociation of catalytic subunits from regulatory subunits.

Protein kinase A (PKA) plays essential roles in diverse cellular functions. However, the spatiotemporal dynamics of endogenous PKA upon activation remain debated. The classical model predicts that PKA catalytic subunits dissociate from regulatory subunits in the presence of cAMP, whereas a second model proposes that catalytic subunits remain associated with regulatory subunits following physiological activation. Here, we report that different PKA subtypes, as defined by the regulatory subunit, exhibit distinct subcellular localization at rest in CA1 neurons of cultured hippocampal slices. Nevertheless, when all tested PKA subtypes are activated by norepinephrine, presumably via the β-adrenergic receptor, catalytic subunits translocate to dendritic spines but regulatory subunits remain unmoved. These differential spatial dynamics between the subunits indicate that at least a significant fraction of PKA dissociates. Furthermore, PKA-dependent regulation of synaptic plasticity and transmission can be supported only by wildtype, dissociable PKA, but not by inseparable PKA. These results indicate that endogenous PKA regulatory and catalytic subunits dissociate to achieve PKA function in neurons.

Synaptopodin: a key regulator of Hebbian plasticity

Synaptopodin, an actin-associated protein found in a subset of dendritic spines in telencephalic neurons, has been described to influence both functional and morphological plasticity under various plasticity paradigms. Synaptopodin is necessary and sufficient for the formation of the spine apparatus, stacks of smooth endoplasmic reticulum cisternae. The spine apparatus is a calcium store that locally regulates calcium dynamics in response to different patterns of activity and is also thought to be a site for local protein synthesis. Synaptopodin is present in ~30% of telencephalic large dendritic spines in vivo and in vitro highlighting the heterogeneous microanatomy and molecular architecture of dendritic spines, an important but not well understood aspect of neuroplasticity. In recent years, it has become increasingly clear that synaptopodin is a formidable regulator of multiple mechanisms essential for learning and memory. In fact, synaptopodin appears to be the decisive factor that determines whether plasticity can occur, acting as a key regulator for synaptic changes. In this review, we summarize the current understanding of synaptopodin’s role in various forms of Hebbian synaptic plasticity.

Simulated synapse loss induces depression-like behaviors in deep reinforcement learning

Deep Reinforcement Learning is a branch of artificial intelligence that uses artificial neural networks to model reward-based learning as it occurs in biological agents. Here we modify a Deep Reinforcement Learning approach by imposing a suppressive effect on the connections between neurons in the artificial network—simulating the effect of dendritic spine loss as observed in major depressive disorder (MDD). Surprisingly, this simulated spine loss is sufficient to induce a variety of MDD-like behaviors in the artificially intelligent agent, including anhedonia, increased temporal discounting, avoidance, and an altered exploration/exploitation balance. Furthermore, simulating alternative and longstanding reward-processing-centric conceptions of MDD (dysfunction of the dopamine system, altered reward discounting, context-dependent learning rates, increased exploration) does not produce the same range of MDD-like behaviors. These results support a conceptual model of MDD as a reduction of brain connectivity (and thus information-processing capacity) rather than an imbalance in monoamines—though the computational model suggests a possible explanation for the dysfunction of dopamine systems in MDD. Reversing the spine-loss effect in our computational MDD model can lead to rescue of rewarding behavior under some conditions. This supports the search for treatments that increase plasticity and synaptogenesis, and the model suggests some implications for their effective administration.

S-ketamine alleviates depression-like behavior and hippocampal neuroplasticity in the offspring of mice that experience prenatal stress

Prenatal stress exerts long-term impact on neurodevelopment in the offspring, with consequences such as increasing the offspring’s risk of depression in adolescence and early adulthood. S-ketamine can produce rapid and robust antidepressant effects, but it is not clear yet whether and how S-ketamine alleviates depression in prenatally stressed offspring. The current study incestigated the preliminary anti-depression mechanism of S-ketamine in prenatally stressed offspring, particularly with regard to neuroplasticity. The pregnant females were given chronic unpredictable mild stress on the 7th-20th day of pregnancy and their male offspring were intraperitoneally injected with a single dose of S-ketamine (10 mg/kg) on postnatal day 42. Our findings showed that S-ketamine treatment counteracted the development of depression-like behaviors in prenatally stressed offspring. At the cellular level, S-ketamine markedly enhanced neuroplasticity in the CA1 hippocampus: Golgi-Cox staining showed that S-ketamine alleviated the reduction of neuronal complexity and dendritic spine density; Transmission electron microscopy indicated that S-ketamine reversed synaptic morphology alterations. At the molecular level, by western blot and RT-PCR we detected that S-ketamine significantly upregulated the expression of BDNF and PSD95 and activated AKT and mTOR in the hippocampus. In conclusion, prenatal stress induced by chronic unpredictable mild stress leads to depressive-like behaviors and hippocampal neuroplasticity impairments in male offspring. S-ketamine can produce antidepressant effects by enhancing hippocampal neuroplasticity via the BDNF/AKT/mTOR signaling pathway.

Atypical antipsychotics improve dendritic spine pathology in temporal lobe cortex neurons in a developmental rodent model of schizophrenia.

"Dendritic spine pathology" refers to alterations in density and morphology of dendritic spines, crucial in corticolimbic neurons in schizophrenia. These structural neuroplasticity changes contribute to the disease's neurobiological underpinnings, alongside alterations in other brain regions, such as temporal lobe cortices like the auditory cortex (Au1) and the entorhinal cortex (Ent), involved in sensory processing, memory, and learning. The neonatal ventral hippocampus lesion (NVHL) in rats exhibits behavioral abnormalities akin to schizophrenia symptoms and corticolimbic dendritic spine pathology, mitigated by atypical antipsychotic drugs (AADs) like risperidone (RISP) and olanzapine (OLZ). This study investigated NVHL-induced dendritic spine pathology in Au1 and Ent, evaluating RISP and OLZ effects. NVHL induced dendritic spine pathology mainly by reducing the dendritic spine density in Au1 and Ent neurons; both RISP and OLZ mitigated it, increasing dendritic spine density and mushroom spine population, the ones related with synaptic strengthening, while decreasing stubby spine population. These findings underscore the role of impaired neuroplasticity in the temporal lobe cortices in schizophrenia pathophysiology and highlight the relevance of the NVHL model for studying neuroplasticity mechanisms in the disease. They also contribute to the growing understanding of targeting structural and functional neuroplasticity for novel drugs in the pharmacotherapy of the disease.

Dynamics of AKAP/Calmodulin complex is largely driven by ionic occupancy state.

AKAP79/150 is a scaffold protein found in dendritic spines and other neuronal compartments. It localizes and regulates phosphorylation by protein kinase A and C and is, in turn regulated by , mediated by Calmodulin (CaM). Thus, the interaction of AKAP79/150 with CaM is of biological interest. A 2017 study used a peptide cross linking coupled to mass spectrometry (XLMS) to identify the CaM binding site on AKAP79/150 and subsequently solved an X-ray crystallography structure of CaM in complex with a short helical AKAP79/150 peptide. The XRD structure revealed an unusual mixed ionic occupancy state of CaM as bound to the AKAP79/150 peptide. In this molecular dynamics-based study, we have explored the motional modes of the CaM-AKAP helix complex under three ionic occupancy conditions. Our results indicate that the dynamics of this CaM backbone is largely dominated by the ionic occupancy state. We find that binding of the AKAP79/150 peptide to CaM is not preferentially stabilized in energetic terms in the Ca2+ state as compared to apo. However, the Mg2+ state is destabilized energetically as compared to the apo state. In addition, in the Ca2+ state, the AKAP79/150 peptide appears to be preferentially stabilized by additional hydrogen bonds. Our simulations suggest that further structural biology studies should be carried out, with a focus on driving the system equilibrium to full occupancy. NMR studies may be able to capture conformational states which are not seen in crystals.

Pretreatment with tetramethylpyrazine alleviated the impairment of learning and memory induced by sevoflurane exposure in neonatal rats

Sevoflurane impairs learning and memory of the developing brain. However, strategies to mitigate these detrimental effects have been scarce. Herein, we investigated whether tetramethylpyrazine could alleviate the impairment of learning and memory and its underlying mechanisim in sevoflurane-exposed neonatal rats. Postnatal 7-day Sprague-Dawley (SD) rats or primary hippocampal neurons were pretreated with tetramethylpyrazine and then exposed to sevoflurane. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and lactate dehydrogenase (LDH) assays were used to detect neuronal injury. Learning and memory function were evaluated by novel object recognition and Morris water maze tests. Long-term potentiation (LTP) was recorded to evaluate synaptic plasticity electrophysiologically in the hippocampal slices. Golgi-Cox staining or PSD95 immunochemistry was used to detect the morphology of dendritic spines. Western blotting was employed to assess the expressions of cleaved Caspase-3, PSD95, NMDAR1, NMDAR2A and NMDAR2B in the hippocampus or cultured neurons. It was found that neonatal exposure of sevoflurane impaired learning and memory, increased neuronal apoptosis, altered the morphology of dendritic spine, upregulated the expressions of NMDAR2A and PSD95, and induced LTP deficits. Pretreatment with tetramethylpyrazine not only alleviated impairment of learning and memory, but also improved sevoflurane-induced changes in neuronal damage, dendritic spine morphology, NMDAR2A and PSD95 expressions, as well as LTP. These findings indicated that pretreatment with tetramethylpyrazine alleviated the impairment of learning and memory induced by sevoflurane through improvement of hippocampal synaptic plasticity in neonatal rats.

Obese male zucker rats show basilar dendritic retraction in the medial prefrontal cortex

Obesity, a prevalent disorder, predisposes individuals to metabolic syndrome, type 2 diabetes mellitus, and high blood pressure. Obesity has been investigated in various organisms that display genetic, high-fat, and high-carbohydrate diet (HFCD)-induced obesity. Recent studies have found that both male and female Zucker rats, which are genetically obese, exhibit alterations in dendritic arborization of neurons in certain structures of the central nervous system. Therefore, the present study aimed to analyze dendritic arborization and dendritic spine density of pyramidal neurons in the medial prefrontal cortex (mPFC) of obese adult male Zucker rats using the Golgi-Cox method and Sholl analysis. Obese male Zucker rats exhibit increased body weight and high concentrations of glucose, triglycerides, and cholesterol. Analysis of mPFC pyramidal neurons in these rats revealed basilar dendritic retraction at a medium distance from the soma, in addition to a reduction in total basilar dendritic length, without any changes in dendritic spine density. These findings are consistent with previous reports, indicating that changes in dendritic retraction may occur as a result of the leptin receptor mutation itself, in addition to the reduction in dendritic arborization observed in other regions of the central nervous system in rats. Furthermore, we suggest the possibility that biological processes modulated by the mPFC, such as foraging and social behavior, are also affected.

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.

Neuronal connectivity, behavioral, and transcriptional alterations associated with the loss of MARK2.

Neuronal connectivity is essential for adaptive brain responses and can be modulated by dendritic spine plasticity and the intrinsic excitability of individual neurons. Dysregulation of these processes can lead to aberrant neuronal activity, which has been associated with numerous neurological disorders including autism, epilepsy, and Alzheimer's disease. Nonetheless, the molecular mechanisms underlying abnormal neuronal connectivity remain unclear. We previously found that the serine/threonine kinase Microtubule Affinity Regulating Kinase 2 (MARK2), also known as Partitioning Defective 1b (Par1b), is important for the formation of dendritic spines invitro. However, despite its genetic association with several neurological disorders, the invivo impact of MARK2 on neuronal connectivity and cognitive functions remains unclear. Here, we demonstrate that the loss of MARK2 invivo results in changes to dendritic spine morphology, which in turn leads to a decrease in excitatory synaptic transmission. Additionally, the loss of MARK2 produces substantial impairments in learning and memory, reduced anxiety, and defective social behavior. Notably, MARK2 deficiency results in heightened seizure susceptibility. Consistent with this observation, electrophysiological analysis of hippocampal slices indicates underlying neuronal hyperexcitability in MARK2-deficient neurons. Finally, RNAseq analysis reveals transcriptional changes in genes regulating synaptic transmission and ion homeostasis. These results underscore the invivo role of MARK2 in governing synaptic connectivity, neuronal excitability, and cognitive functions.

Integration across biophysical scales identifies molecular and cellular correlates of person-to-person variability in human brain connectivity

Brain connectivity arises from interactions across biophysical scales, ranging from molecular to cellular to anatomical to network level. To date, there has been little progress toward integrated analysis across these scales. To bridge this gap, from a unique cohort of 98 individuals, we collected antemortem neuroimaging and genetic data, as well as postmortem dendritic spine morphometric, proteomic and gene expression data from the superior frontal and inferior temporal gyri. Through the integration of the molecular and dendritic spine morphology data, we identified hundreds of proteins that explain interindividual differences in functional connectivity and structural covariation. These proteins are enriched for synaptic structures and functions, energy metabolism and RNA processing. By integrating data at the genetic, molecular, subcellular and tissue levels, we link specific biochemical changes at synapses to connectivity between brain regions. These results demonstrate the feasibility of integrating data from vastly different biophysical scales to provide a more comprehensive understanding of brain connectivity.

Histological features of the Purkinje neurons of the Albino rat (Rattus norvegicus) following letrozole administration.

Aromatase inhibitors are increasingly being used as adjuvant therapy for hormone-responsive cancers. These drugs may reduce the endogenous estrogen production in the cerebellum. Prolonged use has been associated with symptoms such as ataxia, poorer balance performance and diminished verbal memory, suggesting impaired cerebellar function. Thus, this study sought to outline the structural basis for the cerebellar deficits observed. Twenty-seven male rats (3 baseline, 15 experimental, 9 control) aged three months were recruited with the intervention group receiving 0.5 mg/kg of letrozole daily for 50 days by oral gavage while the control group received normal saline. Their cerebella were harvested for histological processing on days 20, 35, and 50. Photomicrographs were taken and analysed using Fiji ImageJ software. The dendritic spine densities and Purkinje linear densities were coded and analyzed using IBM SPSS Statistics version 25.0. A P-value of ≤0.05 was considered significant. A temporal decline in the Purkinje linear density as well as pyknosis and cytoplasmic eosinophilia was noted in the intervention group (P=0.1). Further, the dendritic spine density of the Purkinje neurons in the intervention group was markedly reduced (P=0.01). The reduction in the linear cell density and the dendritic spine density of the Purkinje cells following letrozole administration may provide an anatomical basis for the functional cerebellar deficits seen in chronic aromatase inhibitor use.

Traditional Chinese herbal formula, Fuzi-Lizhong pill, produces antidepressant-like effects in chronic restraint stress mice through systemic pharmacology

Ethnopharmacological relevanceFuzi-Lizhong pill (FLP) is a well-validated traditional Chinese medicine (TCM) formula that has long been used in China for gastrointestinal disease and adjunctive therapy for depression. In our previous study, we reported that the principal herb of FLP, Aconitum carmichaelii Debx. (Fuzi), exhibits antidepressant-like effects. However, there have been no reports on whether FLP produces antidepressant-like effects and its potential molecular mechanisms. Aim of the studyWe aim to demonstrate the antidepressant-like effects of FLP in chronic restraint stress (CRS) mice and to explore the associated molecular mechanisms. Materials and methodsThe active components and probable molecular targets of FLP, as well as the targets related to depression, were identified through network pharmacology. A protein-protein interaction (PPI) network was generated using the overlapping targets, followed by the visualization as well as identification of the core targets associated with the antidepressant-like action of FLP. Subsequently, KEGG and GO enrichment analyses were conducted. UHPLC-MS/MS was employed to further detect the active compounds in FLP. Molecular docking was applied to assess the connections between the active components as well as the core targets. The efficacy of FLP in treating depression and its molecular mechanisms were examined using western blotting, ELISA, 16S rRNA sequencing, HE staining, Nissl staining, and Golgi-Cox staining in a CRS-induced mouse model. ResultsNetwork pharmacology and UHPLC-MS/MS analyses indicated that the active compounds of FLP comprised taraxerol, songorine, neokadsuranic acid B, ginkgetin, hispaglabridin B, quercetin, benzoylmesaconine and liquiritin. KEGG pathway analysis implicated that the PI3K/Akt/mTOR as well as MAPK signaling pathways are closely related to the therapeutic effects of FLP on depression. Molecular docking analysis demonstrated that the main components of FLP bind to PI3K, AKT, mTOR, BDNF and MAPK. FLP significantly decreased immobility in mice that were elevated by CRS in the FST and the TST. FLP also significantly increased sucrose preference in mice after CRS in the SPT. FLP upregulated proteins associated with BDNF-TrkB and PI3K/Akt/mTOR signaling and downregulated proteins associated with MAPK signaling. Serum levels of CORT, IL-6, IL-1β, and TNF-α in CRS mice were significantly decreased following treatment with FLP. In addition, FLP ameliorated CRS-induced gut microbiota dysbiosis as demonstrated by 16S rRNA sequencing analysis. FLP ameliorated CRS-induced intestinal inflammation and neuronal damage. Finally, antidepressant-like effects and concomitant increases in dendritic spine density induced by FLP administration were also reduced after rapamycin treatment. ConclusionThese results demonstrate that FLP has antidepressant-like effects in mice exposed to CRS that involve activation of the PI3K/Akt/mTOR signaling pathway, increase in spinogenesis, inhibition of the MAPK signaling pathway, decrease in inflammation, and amelioration of gut microbiota dysbiosis. These findings provide novel evidence for the clinical application of FLP on depression.