Neuronal Soma Research Articles

Evidence of extraganglionic vagal mechanoreceptors in the mouse vagus nerve.

Vagal afferent neuronal somas are in the nodose and jugular ganglia. In this study, we identified extraganglionic neurons in whole-mount preparations of the vagus nerves from Phox2b-Cre-ZsGreen transgenic mice. These neurons are typically arranged in small clusters and monolayers along the cervical vagus nerve. Although infrequent, these neurons were sometimes observed along the thoracic and esophageal vagus. We performed RNAscope in situ hybridization and confirmed that the extraganglionic neurons detected in this transgenic mouse strain expressed vagal afferent markers (i.e., Phox2b and Slc17a6) as well as markers that identify them as potential gastrointestinal mechanoreceptors (i.e., Tmc3 and Glp1r). We also identified extraganglionic neurons in the vagus nerves of wild-type mice that were injected intraperitoneally with Fluoro-Gold, thereby ruling out possible anatomical discrepancies specific for transgenic mice. In wild-type mice, extraganglionic cells were positive for peripherin, confirming their neuronal nature. Taken together, our findings revealed a previously undiscovered population of extraganglionic neurons associated with the vagus nerve. Going forward, it is important to consider the possible existence of extraganglionic mechanoreceptors that transmit signals from the abdominal viscera in future studies related to vagal structure and function.

Minimally Invasive Microglial and Neuronal Imaging in Mouse Spinal Cord Dorsal Horn

The spinal cord dorsal horn is a relay hub that receives sensory information from the peripheral nervous system and transmits bioelectrical signals to the brain. <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">In vivo</i> imaging of neuronal and glial activity in the dorsal horn can provide insights into both functions and dysfunctions of the neuronal network in the spinal cord. With multimodal NLO microscopy, we identified a thin-myelin sheath region allowing to image over 200 μm deep below pia with subcellular resolution. By using an optically cleared intervertebral window, dorsal horn neuron and microglia activities can be observed without activating spinal cord inflammation. Two-photon imaging of neurons and microglia as well as the optical clearing improvement at different tissue depths were further characterized over time. Using this inflammation-free imaging method, we conducted a longitudinal study of dorsal horn microglia dynamics following sciatic nerve transection. Furthermore, stable <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in vivo</i> calcium imaging of neurons in the dorsal horn was performed with electrical stimulation on the mouse's hind paw. The subcellular-resolution imaging enabled characterization of the distinct calcium transients of neuronal somas and dendrites. This minimally invasive imaging approach to spinal dorsal horn through an optically cleared intervertebral window provides a reliable platform for studying and understanding cellular activities in the spinal dorsal horn.

Optical manipulation of neuronal mitochondria using scanning optical tweezers

Mitochondria play a crucial role in the physiological functions and energy metabolism of neurons, which can help in the understanding of complex biochemical reactions associated with various neurodegenerative diseases. Neurons, being highly differentiated terminal cells, require a greater number of mitochondria than ordinary cells to generate significant amounts of ATP, which is necessary for the growth of differentiated neuronal structures like axons and dendrites and the transmission of electrical signals along neuronal axons. Advancements in imaging technology, electrophysiology, and fluorescence targeting labeling have facilitated the study of mitochondrial movements in neurons and axons. However, disordered mitochondrial movements can hinder their analysis and characterization. Thus, it becomes necessary to artificially control their transport. Here, we demonstrate the utilization of scanning optical tweezers (SOTs) on the stable trapping and precise transport of soma or axon of neurons and enable. The presented method provides an optical approach to the control of mitochondria or other organelles in complex and variable biological environment.

Structurally and Morphologically Distinct Pathological Tau Assemblies Differentially Affect GVB Accumulation.

Tau aggregation is central to the pathogenesis of a large group of neurodegenerative diseases termed tauopathies, but it is still unclear in which way neurons respond to tau pathology and how tau accumulation leads to neurodegeneration. A striking neuron-specific response to tau pathology is presented by granulovacuolar degeneration bodies (GVBs), lysosomal structures that accumulate specific cargo in a dense core. Here we employed different tau aggregation models in primary neurons to investigate which properties of pathological tau assemblies affect GVB accumulation using a combination of confocal microscopy, transmission electron microscopy, and quantitative automated high-content microscopy. Employing GFP-tagged and untagged tau variants that spontaneously form intraneuronal aggregates, we induced pathological tau assemblies with a distinct subcellular localization, morphology, and ultrastructure depending on the presence or absence of the GFP tag. The quantification of the GVB load in the different models showed that an increased GVB accumulation is associated with the untagged tau aggregation model, characterized by shorter and more randomly distributed tau filaments in the neuronal soma. Our data indicate that tau aggregate structure and/or subcellular localization may be key determinants of GVB accumulation.

Effects of Parkin on the Mitochondrial Genome in the Heart and Brain of Mitochondrial Mutator Mice.

Mitochondrial dysfunction has been implicated in neurodegenerative diseases like Parkinson's disease (PD). This study investigates the role of Parkin, a protein involved in mitochondrial quality control, and strongly linked to PD, in the context of mitochondrial DNA (mtDNA) mutations. The authors use mitochondrial mutator mice (PolgD257A/D257A ) (Polg), which are bred with Parkin knockout (PKO) mice or mice with disinhibited Parkin (W402A). In the brain, mtDNA mutations are analyzed in synaptosomes, presynaptic neuronal terminals, which are far from neuronal soma, which likely renders mitochondria there more vulnerable compared with brain homogenate. Surprisingly, PKO results in reduced mtDNA mutations in the brain but increased control region multimer (CRMS) load in synaptosomes. In the heart, both PKO and W402A lead to increased mutations, with W402A showing more mutations in the heart than PKO. Computational analysis reveals many of these mutations are deleterious. These findings suggest that Parkin plays a tissue-dependent role in regulating mtDNA damage response, with differential effects in the brain and heart. Understanding the specific role of Parkin in different tissues may provide insights into the underlying mechanisms of PD and potential therapeutic strategies. Further investigation into these pathways can enhance the understanding of neurodegenerative diseases associated with mitochondrial dysfunction.

The Voltage-Gated Sodium Channel in Drosophila, Para, Localizes to Dendrites As Well As Axons in Mechanosensitive Chordotonal Neurons.

The fruit fly Drosophila melanogaster has provided important insights into how sensory information is transduced by transient receptor potential (TRP) channels in the peripheral nervous system (PNS). However, TRP channels alone have not been able to completely model mechanosensitive transduction in mechanoreceptive chordotonal neurons (CNs). Here, we show that, in addition to TRP channels, the sole voltage-gated sodium channel (NaV) in Drosophila, Para, is localized to the dendrites of CNs. Para is localized to the distal tip of the dendrites in all CNs, from embryos to adults, and is colocalized with the mechanosensitive TRP channels No mechanoreceptor potential C (NompC) and Inactive/Nanchung (Iav/Nan). Para localization also demarcates spike initiation zones (SIZs) in axons and the dendritic localization of Para is indicative of a likely dendritic SIZ in fly CNs. Para is not present in the dendrites of other peripheral sensory neurons. In both multipolar and bipolar neurons in the PNS, Para is present in a proximal region of the axon, comparable to the axonal initial segment (AIS) in vertebrates, 40-60 μm from the soma in multipolar neurons and 20-40 μm in bipolar neurons. Whole-cell reduction of para expression using RNAi in CNs of the adult Johnston's organ (JO) severely affects sound-evoked potentials (SEPs). However, the duality of Para localization in the CN dendrites and axons identifies a need to develop resources to study compartment-specific roles of proteins that will enable us to better understand Para's role in mechanosensitive transduction.

Down regulation of Akt/mTOR signaling pathway proteins in hippocampus of Alzheimer’s disease mouse model

AbstractBackgroundAkt and mechanistic target of rapamycin (mTOR) signaling pathways are implicated in AD pathology. Activation of these pathways by external cues facilitates new protein synthesis at the synapse which leads to the synaptic plasticity. We have demonstrated that activity dependent Akt1 mediated new protein synthesis is abolished in primary cortical neurons of APP/PS1 mice. However, the molecular underpinnings for the impairment of new protein synthesis at the synapse are still elusive. Our aim is to understand the molecular mechanisms prior to manifestation of pathological symptoms by examining the Akt1 and mTOR signaling cascades and new protein synthesis in hippocampus of WT and APP/PS1 male mice.MethodsWe isolated post nuclear supernatant (PNS) and synaptosomes from brain cortices of WT and APP/PS1 mice at different ages. Synaptosomes and PNS were resolved on SDS‐PAGE and immunoblotted against respective antibodies. Further, synaptoneurosomes were also prepared from hippocampus and performed 35S methionine incorporation assay without or with KCl stimulation. Statistical comparisons were performed using two‐way ANOVA followed by Tukey’s post‐hoc test or two‐tailed Mann‐Whitney U test.ResultsWe observed that pAkt1, pGSK3β, pmTOR, pS6 ribosomal protein, p4E‐BP1 levels are significantly decreased in both post nuclear supernatant (PNS) and synaptosomal fractions isolated from hippocampus of one month old (presymptomatic) APP/PS1 compared to WT male mice. Further, activity dependent new protein synthesis at the synapse was significantly impaired in synaptoneurosomes isolated from hippocampus of presymptomatic APP/PS1 mice, and this deficit is sustained at young adults (three months). However, no impairment was observed in basal protein synthesis at both ages in hippocampus of APP/PS1 compared to WT male mice. Down regulation of Akt1 precludes synaptic activity dependent protein translation at the dendrites but not in the soma in hippocampal neurons from APP/PS1 mice compared to WT mice.ConclusionsOur finding demonstrate that the dysregulation of Akt1/mTOR and its downstream signaling molecules in hippocampus contributes to synaptic dysfunction in AD through loss of activity dependent new protein synthesis at the synapse in hippocampus which is essential for synaptic plasticity and maintenance.

Region specific reduction of neurogranin immunostaining in the hippocampus of Alzheimer’s disease brains

AbstractBackgroundNeurogranin (Ng) is a post‐synaptic protein involved in formation of long‐term potentials, and thus plays an important role in learning and memory. The concentration of Ng is increased in the cerebrospinal fluid (CSF) of people with Alzheimer’s disease (AD), and CSF Ng is considered to be a biomarker for synaptic dysfunction in AD. The expression of Ng protein in the human brain and its association with AD pathology, however, are less well studied.MethodUsing immunohistochemical detection methods we investigated the protein expression of Ng, beta‐amyloid (Aβ) and hyperphosphorylated paired helical filament tau (phospho‐tau; ptau) in the post‐mortem hippocampus from 12 AD patients and 12 cognitively unaffected controls (NC). We compared the number of positively stained cells (Ng, ptau) or plaques (Aβ) in the hippocampal sub‐regions (dentate gyrus, Cornu Ammonis (CA) fields 1‐3) from the 2 groups. Donor samples with intermediate AD (i.e., Braak stage III‐IV) or NC (Braak 0‐I/II) were analyzed using Image Pro Premier automated image analysis software (version 10.01). The data were normalized to the number of cells or clusters/mm2 and analyzed using a one‐way ANOVA. Using a test of circularity (Cir = (4*A)/Pi*MaxFeret2), we further investigated morphological differences in the Ng positive neurons by measuring the Ng expression in the apical dendrite of pyramidal neurons.ResultThere was a 40% reduction of Ng immunopositive neurons in AD compared to NC in the CA3(N = 24;p = 0.0003) and in the CA1(N = 24;p = 0.014) hippocampal sub‐regions. Ng positive cells did not change significantly in AD compared to NC in the other hippocampal regions. Additionally, we showed that CA1 region, Ng was localized mainly in the neuronal cell soma of AD donors, while its expression extended into the apical dendrites of NC donors (N = 24;p = 0.006). In AD, Ng immunostaining negatively correlated with ptau immunopositive neurofibrillary tangles (r = ‐0.70;p = 0.04)ConclusionSelective loss of Ng immunostaining in certain subregions of the hippocampus suggests that synaptic damage in AD maybe a region‐specific event. The mechanism underlying such regional synaptic damage and the changes in Ng expression will be key in understanding the selective vulnerability of brain regions to AD.

Stabilization of 14-3-3 protein-protein interactions with Fusicoccin-A decreases alpha-synuclein dependent cell-autonomous death in neuronal and mouse models

Synucleinopathies are a group of neurodegenerative diseases without effective treatment characterized by the abnormal aggregation of alpha-synuclein (aSyn) protein. Changes in levels or in the amino acid sequence of aSyn (by duplication/triplication of the aSyn gene or point mutations in the encoding region) cause familial cases of synucleinopathies. However, the specific molecular mechanisms of aSyn-dependent toxicity remain unclear. Increased aSyn protein levels or pathological mutations may favor abnormal protein-protein interactions (PPIs) that could either promote neuronal death or belong to a coping response program against neurotoxicity. Therefore, the identification and modulation of aSyn-dependent PPIs can provide new therapeutic targets for these diseases. To identify aSyn-dependent PPIs we performed a proximity biotinylation assay based on the promiscuous biotinylase BioID2. When expressed as a fusion protein, BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by streptavidin affinity purification and mass spectrometry. The aSyn interactome was analyzed using BioID2-tagged wild-type (WT) and pathological mutant E46K aSyn versions in HEK293 cells. We found the 14-3-3 epsilon isoform as a common protein interactor for WT and E46K aSyn. 14‐3-3 epsilon correlates with aSyn protein levels in brain regions of a transgenic mouse model overexpressing WT human aSyn. Using a neuronal model in which aSyn cell-autonomous toxicity is quantitatively scored by longitudinal survival analysis, we found that stabilization of 14‐3-3 protein-proteins interactions with Fusicoccin-A (FC-A) decreases aSyn-dependent toxicity. Furthermore, FC-A treatment protects dopaminergic neuronal somas in the substantia nigra of a Parkinson's disease mouse model. Based on these results, we propose that the stabilization of 14‐3-3 epsilon interaction with aSyn might reduce aSyn toxicity, and highlight FC-A as a potential therapeutic compound for synucleinopathies.

Role of potassium ion diffusion from neurons to capillaries in neurovascular coupling: effects of astrocytic endfeet geometry

Neurovascular coupling (NVC) refers to a local increase in cerebral blood flow (CBF) in response to increased neuronal activity. Diffusion of K + released by neurons through the extracellular space (ECS) to capillaries may contribute to NVC. Increased perivascular K + levels can initiate a conducted response that travels to arterioles, triggering vasodilation and increased CBF. Astrocytic endfeet almost completely cover cerebral capillaries, and K + must diffuse through narrow clefts between endfeet to reach the capillaries. It has therefore been assumed that astrocytes form a barrier to diffusion of K + in the ECS. The goal of this study is to determine the effects of astrocytic endfeet on the diffusion of K + from neurons to blood vessels. Two simplified 2D geometries of the ECS, endfeet, and perivascular space (PVS) are used. The first geometry represents diffusion of K + from a source 1 μm away from a capillary of diameter 5 μm. The second geometry represents K + diffusion from a neuron 15 μm from the capillary, the average distance between a neuronal soma and the nearest blood vessel. A baseline [K + ] of 3 mM is assumed throughout the region. K + release from neurons is modeled as a prescribed concentration of 12 mM along the outer boundary of the ECS. The time-dependent diffusion equation is used to describe K + diffusion through the region. Effective diffusivity of K + is 937.5 μm 2 /s. Standard conditions are a PVS width of 100 nm and an astrocytic endfeet cleft width of 20 nm, with 2.5 clefts per vessel circumference such that the total width of the clefts represents 0.31% of the capillary’s circumference. In the first geometry, [K + ] in the PVS approaches 12 mM within 0.05 s. Decreasing the astrocytic endfeet cleft width to 6.67 nm slows the rise in [K + ] to 10 mM at 0.05 s. Increasing PVS width also slows the rise in [K + ]. In the second geometry, the rise in [K + ] is slower due to the greater diffusion distance and PVS [K + ] approaches 12 mM within 0.5 s. Decreasing cleft width or increasing PVS width in the second geometry slightly slows the rise in [K + ]. The predicted rise in [K + ] at the blood vessel occurs well within the time that increased CBF is observed in NVC, 1-2 seconds. The predicted PVS [K + ] levels are sufficient to cause the vasodilation seen in NVC. The present results suggest that direct diffusion of K + through the ECS is a possible NVC signaling mechanism. Supported by NIH Grant U01 HL133362. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activation of mTOR signaling in the paraventricular nucleus of the hypothalamus (PVN) contributes to stress- and BDNF-induced hypertensive responses

The paraventricular nucleus of the hypothalamus (PVN) integrates afferent inputs from limbic, medullary and hypothalamic regions to orchestrate cardiovascular stress responses via its medullary and spinal projections. Brain-derived neurotrophic factor (BDNF) is an important pro-hypertensive regulator in the PVN upregulated during stress that transforms the PVN neurocircuitry to facilitate responsiveness to hypertensive stimuli by shifting the balance of excitatory/inhibitory synaptic mechanisms. Mechanistic target of rapamycin (mTOR), as part of mTOR complex-1 (mTORC1), is stimulated by trophic factors, such as BDNF, in other brain regions to induce changes in neuronal morphology and increases in neuronal excitability. We tested the hypothesis that activation of mTORC1 in the PVN by BDNF and acute stress plays a key role in stimulating hypertensive responses. Male Sprague Dawley (SD) rats (n=4/group) received bilateral PVN injections of AAV2 viral vectors expressing myc-tagged BDNF (BDNFmyc), shRNA against TSC1 (a repressor of mTORC1) to stimulate mTORC1, shRNA against Raptor (a key component of the mTORC1) to inhibit mTORC1 or GFP for control. Blood pressure and heart rate were monitored by telemetry for 4 wks, and cardiovascular responses to restraint (60min) and water stress (15 minutes, 1-cm deep, 25°C water) were recorded 4-5 wks after vector injections. Vector injections were confirmed and neuronal morphology as well as pS6 levels (a marker of mTOR activity) were analyzed with immunofluorescence. Activation mTORC1 with TSC1 knock-down significantly increased resting blood pressure compared with control, but inhibition of mTORC1 with Raptor knock-down had no effect. BDNF overexpression significantly elevated blood pressure, as we have shown previously, and these increases were completely abolished by Raptor knock-down (daytime mean arterial pressure: shTSC1: 108.0±1.4*; BDNFmyc: 131.4±2.5*; shRaptor: 101.8±0.9; BDNF+shRaptor: 102.7±2.8#, GFP: 96.5±1.8 mmHg at week 4, *p&lt;0.05 vs GFP; #p&lt;0.05 vs. BDNFmyc). In addition, mTORC1 inhibition with Raptor knock-down also significantly diminished stress-induced hypertensive responses. Maximum blood pressure increase in shRaptor rats was 30.3±1.3 mmHg during restraint and 26.0±3.4 mmHg during water stress vs 43.8±2.2 and 37.4±2.5 mmHg in control rats (p&lt;0.05). Immunofluorescence of pS6, an indicator of mTOR activity, and neuronal soma size were significantly elevated by both TSC1 knock-down and BDNF overexpression in the PVN, whereas Raptor knock-down reduced pS6 intensity and soma size and completely abolished the effects of BDNF. In summary, we demonstrated that mTOR activation in the PVN significantly increased blood pressure both during baseline conditions and during acute stress, and mediated hypertensive responses and neuronal morphological changes elicited by BDNF. Supported by R01 HL133211. R01 HL133211 (BE), R03 AG072016 (BE) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Immature neurons in the primate amygdala: Changes with early development and disrupted early environment

In human and nonhuman primates, the amygdala paralaminar nucleus (PL) contains immature neurons. To explore the PL’s potential for cellular growth during development, we compared PL neurons in (1) infant and adolescent macaques (control, maternally-reared), and in (2) infant macaques that experienced separation from their mother in the first month of life compared to control maternally-reared infants. In maternally-reared animals, the adolescent PL had fewer immature neurons, more mature neurons, and larger immature soma volumes compared to infant PL. There were also fewer total neurons (immature plus mature) in adolescent versus infant PL, suggesting that some neurons move out of the PL by adolescence. Maternal separation did not change mean immature or mature neuron counts in infant PL. However, across all infant animals, immature neuron soma volume was strongly correlated with mature neuron counts. TBR1 mRNA, a transcript required for glutamatergic neuron maturation, is significantly reduced in the maternally-separated infant PL (DeCampo et al., 2017), and was also positively correlated with mature neuron counts in infant PL. We conclude that immature neurons gradually mature by adolescence, and that the stress of maternal separation may shift this trajectory, as revealed by correlations between TBR1 mRNA and mature neuron numbers across animals.

Postmortem Brains from Subjects with Diabetes Mellitus Display Reduced GLUT4 Expression and Soma Area in Hippocampal Neurons: Potential Involvement of Inflammation.

Diabetes mellitus (DM) is an important risk factor for dementia, which is a common neurodegenerative disorder. DM is known to activate inflammation, oxidative stress, and advanced glycation end products (AGEs) generation, all capable of inducing neuronal dysfunctions, thus participating in the neurodegeneration progress. In that process, disturbed neuronal glucose supply plays a key role, which in hippocampal neurons is controlled by the insulin-sensitive glucose transporter type 4 (GLUT4). We investigated the expression of GLUT4, nuclear factor NF-kappa B subunit p65 [NFKB (p65)], carboxymethyllysine and synapsin1 (immunohistochemistry), and soma area in human postmortem hippocampal samples from control, obese, and obese+DM subjects (41 subjects). Moreover, in human SH-SY5Y neurons, tumor necrosis factor (TNF) and glycated albumin (GA) effects were investigated in GLUT4, synapsin-1 (SYN1), tyrosine hydroxylase (TH), synaptophysin (SYP) proteins, and respective genes; NFKB binding activity in the SLC2A4 promoter; effects of increased histone acetylation grade by histone deacetylase 3 (HDAC3) inhibition. Hippocampal neurons (CA4 area) of obese+DM subjects displayed reduced GLUT4 expression and neuronal soma area, associated with increased expression of NFKB (p65). Challenges with TNF and GA decreased the SLC2A4/GLUT4 expression in SH-SY5Y neurons. TNF decreased SYN1, TH, and SYP mRNAs and respective proteins, and increased NFKB binding activity in the SLC2A4 promoter. Inhibition of HDAC3 increased the SLC2A4 expression and the total neuronal content of CRE-binding proteins (CREB/ICER), and also counterbalanced the repressor effect of TNF upon these parameters. This study revealed reduced postmortem human hippocampal GLUT4 content and neuronal soma area accompanied by increased proinflammatory activity in the brains of DM subjects. In isolated human neurons, inflammatory activation by TNF reduced not only the SLC2A4/GLUT4 expression but also the expression of some genes related to neuronal function (SYN1, TH, SYP). These effects may be related to epigenetic regulations (H3Kac and H4Kac status) since they can be counterbalanced by inhibiting HDAC3. These results uncover the improvement in GLUT4 expression and/or the inhibition of HDAC3 as promising therapeutic targets to fight DM-related neurodegeneration.

The role of orexin neuron activity in sleep/wakefulness regulation

Orexin (also known as hypocretin) is a neuropeptide exclusively synthesized in the neurons of the lateral hypothalamus (LH). Initially orexin was thought to be involved in the regulation of feeding behavior. However, it is now known to also be a critical regulator of sleep/wakefulness, especially the maintenance of wakefulness. Although the somas of orexin neurons are exclusively located in the LH, these neurons send axons throughout the brain and spinal cord. Orexin neurons integrate inputs from various brain regions and project to neurons that are involved in the regulation of sleep/wakefulness. Orexin knockout mice have a fragmentation of sleep/wakefulness and cataplexy-like behavior arrest, which is similar to the sleep disorder narcolepsy. Recent progress with manipulation of neural activity of targeted neurons, using experimental tools such as optogenetics and chemogenetics, has emphasized the role of orexin neuron activity on the regulation of sleep/wakefulness. Recording of orexin neuron activity in vivo using electrophysiological and gene-encoded calcium indicator proteins revealed that these cells have specific activity patterns across sleep/wakefulness state changes. Here, we also discuss not only the role of the orexin peptide, but also the role of other co-transmitters that are synthesized and released from orexin neurons and involved in sleep/wakefulness regulation.

Neonatal sevoflurane exposure induces long-term changes in dendritic morphology in juvenile rats and mice.

General anesthetics are potent neurotoxins when given during early development, causing apoptotic deletion of substantial number of neurons and persistent neurocognitive and behavioral deficits in animals and humans. The period of intense synaptogenesis coincides with the peak of susceptibility to deleterious effects of anesthetics, a phenomenon particularly pronounced in vulnerable brain regions such as subiculum. With steadily accumulating evidence confirming that clinical doses and durations of anesthetics may permanently alter the physiological trajectory of brain development, we set out to investigate the long-term consequences on dendritic morphology of subicular pyramidal neurons and expression on genes regulating the complex neural processes such as neuronal connectivity, learning, and memory. Using a well-established model of anesthetic neurotoxicity in rats and mice neonatally exposed to sevoflurane, a volatile general anesthetic commonly used in pediatric anesthesia, we report that a single 6 h of continuous anesthesia administered at postnatal day (PND) 7 resulted in lasting dysregulation in subicular mRNA levels of cAMP responsive element modulator (Crem), cAMP responsive element-binding protein 1 (Creb1), and Protein phosphatase 3 catalytic subunit alpha, a subunit of calcineurin (Ppp3ca) (calcineurin) when examined during juvenile period at PND28. Given the critical role of these genes in synaptic development and neuronal plasticity, we deployed a set of histological measurements to investigate the implications of anesthesia-induced dysregulation of gene expression on morphology and complexity of surviving subicular pyramidal neurons. Our results indicate that neonatal exposure to sevoflurane induced lasting rearrangement of subicular dendrites, resulting in higher orders of complexity and increased branching with no significant effects on the soma of pyramidal neurons. Correspondingly, changes in dendritic complexity were paralleled by the increased spine density on apical dendrites, further highlighting the scope of anesthesia-induced dysregulation of synaptic development. We conclude that neonatal sevoflurane induced persistent genetic and morphological dysregulation in juvenile rodents, which could indicate heightened susceptibility toward cognitive and behavioral disorders we are beginning to recognize as sequelae of early-in-life anesthesia.

Astrotactin 2 (ASTN2) regulates emotional and cognitive functions by affecting neuronal morphogenesis and monoaminergic systems.

Astrotactin2 (ASTN2) regulates neuronal migration and synaptic strength through the trafficking and degradation of surface proteins. Deletion of ASTN2 in copy number variants has been identified in patients with schizophrenia, bipolar disorder, and autism spectrum disorder in copy number variant (CNV) analysis. Disruption of ASTN2 is a risk factor for these neurodevelopmental disorders, including schizophrenia, bipolar disorder, autism spectrum disorder, and attention deficit hyperactivity disorder. However, the importance of ASTN2 in physiological functions remains poorly understood. To elucidate the physiological functions of ASTN2, we investigated whether deficiency of ASTN2 affects cognitive and/or emotional behaviors and neurotransmissions using ASTN2-deficient mice. Astn2 knockout (KO) mice produced by CRISPR/Cas9 technique showed no obvious differences in physical characteristics and circadian rhythm. Astn2 KO mice showed increased exploratory activity in a novel environment, social behavior and impulsivity, or decreased despair-, anxiety-like behaviors and exploratory preference for the novel object. Some behavioral abnormalities, such as increased exploratory activity and impulsivity, or decreased exploratory preference were specifically attenuated by risperidone, but not by haloperidol. While, the both drugs did not affect any emotion-related behavioral abnormalities in Astn2 KO mice. Dopamine contents were decreased in the striatum, and serotonin or dopamine turnover were increased in the striatum, nucleus accumbens, and amygdala of Astn2 KO mice. In morphological analyses, thinning of neural cell layers in the hippocampus, reduction of neural cell bodies in the prefrontal cortex, and decrease in spine density and PSD95 protein in both tissues were observed in Astn2 KO mice. The present findings suggest that ASTN2 deficiency develops some emotional or cognitive impairments related to monoaminergic dysfunctions and abnormal neuronal morphogenesis with shrinkage of neuronal soma. ASTN2 protein may contribute to the pathogenic mechanism and symptom onset of mental disorders.

Blockade of orexin receptor 1 attenuates morphine protracted abstinence-induced anxiety-like behaviors in male mice

One negative emotional state from morphine protracted abstinence is anxiety which can drive craving and relapse risk in opioid addicts. Although the orexinergic system has been reported to be important in mediating emotion processing and addiction, the role of orexinergic system in anxiety from drug protracted abstinence remains elusive. In this study, by using behavioral test, western blot, electrophysiology and virus-mediated regulation of orexin receptor 1 (OX1R), we found that: (1) Intraperitoneal and intra-VTA administration of a selective OX1R antagonist SB334867 alleviated anxiety-like behaviors in open field test (OFT) but not in elevated plus maze test (EPM) in morphine protracted abstinent male mice. (2) OX1R expression in the VTA was upregulated by morphine withdrawal. (3) Virus-mediated knockdown of OX1R in the VTA prevented morphine abstinence-induced anxiety-like behaviors and virus-mediated overexpression of OX1R in the VTA was sufficient to produce anxiety-like behaviors in male mice. (4) The VTA neuronal activity was increased significantly induced by morphine protracted abstinence, which was mediated by OX1R. (5) OX1R was widely distributed in the neuronal soma and processes of dopaminergic and non-dopaminergic neurons in the VTA. The findings revealed that the OX1R mediates morphine abstinence-induced anxiety-like behaviors and the VTA plays a critical role in this effect.

A 3D human co-culture to model neuron-astrocyte interactions in tauopathies

BackgroundIntraneuronal tau aggregation is the major pathological hallmark of neurodegenerative tauopathies. It is now generally acknowledged that tau aggregation also affects astrocytes in a cell non-autonomous manner. However, mechanisms involved are unclear, partly because of the lack of models that reflect the situation in the human tauopathy brain. To accurately model neuron-astrocyte interaction in tauopathies, there is a need for a model that contains both human neurons and human astrocytes, intraneuronal tau pathology and mimics the three-dimensional architecture of the brain.ResultsHere we established a novel 100–200 µm thick 3D human neuron/astrocyte co-culture model of tau pathology, comprising homogenous populations of hiPSC-derived neurons and primary human astrocytes in microwell format. Using confocal, electron and live microscopy, we validate the procedures by showing that neurons in the 3D co-culture form pre- and postsynapses and display spontaneous calcium transients within 4 weeks. Astrocytes in the 3D co-culture display bipolar and stellate morphologies with extensive processes that ensheath neuronal somas, spatially align with axons and dendrites and can be found perisynaptically. The complex morphology of astrocytes and the interaction with neurons in the 3D co-culture mirrors that in the human brain, indicating the model’s potential to study physiological and pathological neuron-astrocyte interaction in vitro. Finally, we successfully implemented a methodology to introduce seed-independent intraneuronal tau aggregation in the 3D co-culture, enabling study of neuron-astrocyte interaction in early tau pathogenesis.ConclusionsAltogether, these data provide proof-of-concept for the utility of this rapid, miniaturized, and standardized 3D model for cell type-specific manipulations, such as the intraneuronal pathology that is associated with neurodegenerative disorders.

Paclitaxel effects on axonal localization and vesicular trafficking of NaV1.8.

Patients treated with paclitaxel (PTX) or other antineoplastic agents can experience chemotherapy-induced peripheral neuropathy (CIPN), a debilitating side effect characterized by numbness and pain. PTX interferes with microtubule-based transport, which inhibits tumor growth via cell cycle arrest but can also affect other cellular functions including trafficking of ion channels critical to transduction of stimuli by sensory neurons of the dorsal root ganglia (DRG). We examined the effects of PTX on voltage-gated sodium channel NaV1.8, which is preferentially expressed in DRG neurons, using a microfluidic chamber culture system and chemigenetic labeling to observe anterograde channel transport to the endings of DRG axons in real time. PTX treatment increased the numbers of NaV1.8-containing vesicles traversing the axons. Vesicles in PTX-treated cells exhibited greater average velocity, along with shorter and less frequent pauses along their trajectories. These events were paralleled by greater surface accumulation of NaV1.8 channels at the distal ends of DRG axons. These results were consistent with observations that NaV1.8 is trafficked in the same vesicles containing NaV1.7 channels, which are also involved in pain syndromes in humans and are similarly affected by PTX treatment. However, unlike Nav1.7, we did not detect increased NaV1.8 current density measured at the neuronal soma, suggesting a differential effect of PTX on trafficking of NaV1.8 in soma versus axonal compartments. Therapeutic targeting of axonal vesicular traffic would affect both Nav1.7 and Nav1.8 channels and increase the possibilities of alleviating pain associated with CIPN.

Study of Effector CD8+ T Cell Interactions with Cortical Neurons in Response to Inflammation in Mouse Brain Slices and Neuronal Cultures

Cytotoxic CD8+ T cells contribute to neuronal damage in inflammatory and degenerative CNS disorders, such as multiple sclerosis (MS). The mechanism of cortical damage associated with CD8+ T cells is not well understood. We developed in vitro cell culture and ex vivo brain slice co-culture models of brain inflammation to study CD8+ T cell-neuron interactions. To induce inflammation, we applied T cell conditioned media, which contains a variety of cytokines, during CD8+ T cell polyclonal activation. Release of IFNγ and TNFα from co-cultures was verified by ELISA, confirming an inflammatory response. We also visualized the physical interactions between CD8+ T cells and cortical neurons using live-cell confocal imaging. The imaging revealed that T cells reduced their migration velocity and changed their migratory patterns under inflammatory conditions. CD8+ T cells increased their dwell time at neuronal soma and dendrites in response to added cytokines. These changes were seen in both the in vitro and ex vivo models. The results confirm that these in vitro and ex vivo models provide promising platforms for the study of the molecular details of neuron-immune cell interactions under inflammatory conditions, which allow high-resolution live microscopy and are readily amenable to experimental manipulation.