Brain-wide neuronal circuit connectome of human glioblastoma.

Glioblastoma (GBM), a universally fatal brain cancer, infiltrates the brain and can be synaptically innervated by neurons, which drives tumor progression 1-6 . Synaptic inputs onto GBM cells identified so far are largely short-range and glutamatergic 7-9 . The extent of integration of GBM cells into brain-wide neuronal circuitry is not well understood. Here we applied a rabies virus-mediated retrograde monosynaptic tracing approach 10-12 to systematically investigate circuit integration of human GBM organoids transplanted into adult mice. We found that GBM cells from multiple patients rapidly integrated into brain-wide neuronal circuits and exhibited diverse local and long-range connectivity. Beyond glutamatergic inputs, we identified a variety of neuromodulatory inputs across the brain, including cholinergic inputs from the basal forebrain. Acute acetylcholine stimulation induced sustained calcium oscillations and long-lasting transcriptional reprogramming of GBM cells into a more invasive state via the metabotropic CHRM3 receptor. CHRM3 downregulation suppressed GBM cell invasion, proliferation, and survival in vitro and in vivo. Together, these results reveal the capacity of human GBM cells to rapidly and robustly integrate into anatomically and molecularly diverse neuronal circuitry in the adult brain and support a model wherein rapid synapse formation onto GBM cells and transient activation of upstream neurons may lead to a long-lasting increase in fitness to promote tumor infiltration and progression.

fMRI, LFP, and anatomical evidence for hierarchical nociceptive routing pathway between somatosensory and insular cortices.

The directional organization of multiple nociceptive regions, particularly within obscure operculoinsular areas, underlying multidimensional pain processing remains elusive. This study aims to establish the fundamental organization between somatosensory and insular cortices in routing nociceptive information. By employing an integrated multimodal approach of high-field fMRI, intracranial electrophysiology, and transsynaptic viral tracing in rats, we observed a hierarchically organized connection of S1/S2 → posterior insula → anterior insula in routing nociceptive information. The directional nociceptive pathway determined by early fMRI responses was consistent with that examined by early evoked LFP, intrinsic effective connectivity, and anatomical projection, suggesting fMRI could provide a valuable facility to discern directional neural circuits in animals and humans non-invasively. Moreover, our knowledge of the nociceptive hierarchical organization of somatosensory and insular cortices and the interface role of the posterior insula may have implications for the development of targeted pain therapies.

Pleiotropic immunoregulation by growth-blocking peptide in Ostrinia furnacalis.

Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth-blocking peptide (GBP) is a member of the stress-responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity-related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens.

Cerebellar Purkinje cell firing promotes conscious recovery from anesthesia state through coordinating neuronal communications with motor cortex.

Background: The neurobiological basis of gaining consciousness from unconscious state induced by anesthetics remains unknown. This study was designed to investigate the involvement of the cerebello-thalamus-motor cortical loop mediating consciousness transitions from the loss of consciousness (LOC) induced by an inhalational anesthetic sevoflurane in mice. Methods: The neural tracing and fMRI together with opto-chemogenetic manipulation were used to investigate the potential link among cerebello-thalamus-motor cortical brain regions. The fiber photometry of calcium and neurotransmitters, including glutamate (Glu), γ-aminobutyric acid (GABA) and norepinephrine (NE), were monitored from the motor cortex (M1) and the 5th lobule of the cerebellar vermis (5Cb) during unconsciousness induced by sevoflurane and gaining consciousness after sevoflurane exposure. Cerebellar Purkinje cells were optogenetically manipulated to investigate their influence on consciousness transitions during and after sevoflurane exposure. Results: Activation of 5Cb Purkinje cells increased the Ca2+ flux in the M1 CaMKIIα+ neurons, but this increment was significantly reduced by inactivation of posterior and parafascicular thalamic nucleus. The 5Cb and M1 exhibited concerted calcium flux, and glutamate and GABA release during transitions from wakefulness, loss of consciousness, burst suppression to conscious recovery. Ca2+ flux and Glu release in the M1, but not in the 5Cb, showed a strong synchronization with the EEG burst suppression, particularly, in the gamma-band range. In contrast, the Glu, GABA and NE release and Ca2+ oscillations were coherent with the EEG gamma band activity only in the 5Cb during the pre-recovery of consciousness period. The optogenetic activation of Purkinje cells during burst suppression significantly facilitated emergence from anesthesia while the optogenetic inhibition prolonged the time to gaining consciousness. Conclusions: Our data indicate that cerebellar neuronal communication integrated with motor cortex through thalamus promotes consciousness recovery from anesthesia which may likely serve as arousal regulation.

Identification and characteristic analysis of an extracellular signal-regulated kinase from Ostrinia furnacalis Guenée.

The extracellular signal-regulated kinase (ERK) pathway, a critical genetic determinant, controls diverse physiological functions, including innate immunity, development, and stress response. In the current study, a full-length cDNA (1592bp) encoding the ERK gene (OfERK) was cloned from Ostrinia furnacalis Guenée (GenBank accession number: MF797866). The open reading frame of the OfERK gene encoded 364 amino acids and shared 96.43%-98.08% amino acid identities with other insect mitogen-activated protein kinases. For spatiotemporal analysis of the expression pattern, OfERK exhibited a significant peak expression on the 3rd day of the pupa stage and showed the highest expression in hemocytes specifically. Indirect immunofluorescence assays and immuno-electron microscopy revealed a wide distribution of the OfERK protein in hemocytes and epidermis. Moreover, the results demonstrated that the Bt Cry1Ab-activated toxin significantly induces the expression of OfERK. Other genes related to immune response, development, and stress response exhibited dynamic changes in expression after Cry1Ab oral treatment. The expression of OfERK was downregulated through RNA interference, and the correlation of its expression with other related genes was verified using quantitative real-time polymerase chain reaction. Our study provides valuable insights into the regulatory mechanism of ERK in insects for future studies.

Natural bioactive lysosomes extracted from multiple cells for tumor therapy.

Nanoparticles of biological origin exhibit many unique properties in biological applications due to their exquisite structure, specific composition, and natural biological functionality. In this study, we obtained lysosomes from three distinct cell types (one normal cell and two activated immune cells) and demonstrated their potential as natural therapeutic nanoparticles for tumor therapy. In vitro experiments revealed that these lysosomes maintained their structural integrity, were well-distributed, and exhibited significant biological activity, which effectively induced cancer cell death by generating ROS and disrupting biological substrates. Additionally, in vivo investigations showed that these lysosomes could accumulate in tumor tissues after intravenous administration and exhibited exceptional therapeutic effects through the destruction of tumor blood vessels and the degradation of immunosuppressive proteins, with complete tumor disappearance in a single treatment. This research on the utilization of bioactive lysosomes for tumor treatment provides valuable insights into drug development and tumor treatment, particularly when conventional approaches have proven ineffective.

The analysis of HPV integration sites based on nanopore sequencing and the profiling changes along the course of photodynamic therapy.

OBJECTIVE: To detect the HPV genotype and integration sites in patients with high-risk HPV infection at different stages of photodynamic therapy using nanopore technology and to evaluate the treatment effect. METHODS: Four patients with HPV infection were selected and subjected to photodynamic therapy, and cervical exfoliated cell was sampled at before treatment, after three courses of treatment and six courses of treatment, their viral abundance and insertion sites were analyzed by nanopore technology, and pathological examinations were performed before and after treatment. In this study, we developed a novel assay that combined viral sequence enrichment and Nanopore sequencing for identification of HPV genotype and integration sites at once. The assay has obvious advantages over qPCR or NGS-based methods, as it has better sensitivity after viral sequences enrichment and can generate long-reads (kb to Mb) for better detection rate of structure variations, moreover, fast turn-around time for real-time viral sequencing and analysis. RESULTS: The pathological grade was reduced in all four patients after photodynamic therapy. Virus has been cleared in two cases after treatment, the virus amount reduced after treatment but not completely cleared in one case, and two type viruses were cleared and one type virus persisted after treatment in the last patient with multiple infection. Viral abundance and the number of integration sites were positively correlated. Gene enrichment analysis showed complete viral clearance in 1 patient and 3 patients required follow-up. CONCLUSION: Nanopore sequencing can effectively monitor the abundance of HPV viruses and integration sites to show the presence status of viruses, and combined with the results of gene enrichment analysis, the treatment effect can be dynamically assessed.

Developing a trans-multisynaptic tracer to map the neural circuit of recovered sciatic nerve after treatment with nerve growth factor.

Nerve growth factor (NGF) has been shown to support the survival and differentiation of neurons. In this study, we first developed a retrograde trans-multisynaptic tracer PRV580 expressing the mCherry fluorescent protein based on pseudorabies virus Bartha strain to map the neural circuit of sciatic nerve. Secondly, the newly developed PRV580 was used to map the neural circuit of the recovering sciatic nerve upon treatment with NGF. Our results showed that red signals from PRV580 were observed in various brain regions. Among these regions, many areas of the pyramidal system and the extra-pyramidal system had been mapped, accounting for as much as 56.8 % of the total inputs. Furthermore, we found that NGF could significantly increase the ratio of total input (29.05 %) compared to PBS (3.65 %), indicating that NGF indeed can aid in the repair of injured sciatic nerve. These findings indicated that NGF has therapeutic ability for the treatment of peripheral nerve injuries and virus-based tracers can be used to monitor the recovery.

Oncolytic Viral Therapy for Glioma by Recombinant Sindbis Virus.

BACKGROUND: The characteristics of glioblastoma, such as drug resistance during treatment, short patient survival, and high recurrence rates, have made patients with glioblastoma more likely to benefit from oncolytic therapy. METHODS: In this study, we investigated the safety of the sindbis virus by injecting virus intravenously and intracranially in mice and evaluated the therapeutic effect of the virus carrying different combinations of IL-12, IL-7, and GM-CSF on glioma in a glioma-bearing mouse model. RESULTS: SINV was autologously eliminated from the serum and organs as well as from neural networks after entering mice. Furthermore, SINV was restricted to the injection site in the tree shrew brain and did not spread throughout the whole brain. In addition, we found that SINV-induced apoptosis in conjunction with the stimulation of the immune system by tumor-killing cytokines substantially suppressed tumor development. It is worth mentioning that SINV carrying IL-7 and IL-12 had the most notable glioma-killing effect. Furthermore, in an intracranial glioma model, SINV containing IL-7 and IL-12 effectively prolonged the survival time of mice and inhibited glioma progression. CONCLUSIONS: These results suggest that SINV has a significant safety profile as an oncolytic virus and that combining SINV with cytokines is an efficient treatment option for malignant gliomas.

Paraventricular Hypothalamic Nucleus Upregulates Intraocular Pressure Via Glutamatergic Neurons.

Purpose: The neuroregulatory center of intraocular pressure (IOP) is located in the hypothalamus. An efferent neural pathway exists between the hypothalamic nuclei and the autonomic nerve endings in the anterior chamber of the eye. This study was designed to investigate whether the paraventricular hypothalamic nucleus (PVH) regulates IOP as the other nuclei do. Methods: Optogenetic manipulation of PVH neurons was used in this study. Light stimulation was applied via an optical fiber embedded over the PVH to activate projection neurons after AAV2/9-CaMKIIα-hChR2-mCherry was injected into the right PVH of C57BL/6J mice. The same methods were used to inhibit projection neurons after AAV2/9-CaMKIIα-eNpHR3.0-mCherry was injected into the bilateral PVH of C57BL/6J mice. AAV2/9-EF1α-DIO-hChR2-mCherry was injected into the right PVH of Vglut2-Cre mice to elucidate the effect of glutamatergic neuron-specific activation. IOP was measured before and after light manipulation. Associated nuclei activation was clarified by c-Fos immunohistochemical staining. Only mice with accurate viral expression and fiber embedding were included in the statistical analysis. Results: Activation of projection neurons in the right PVH induced significant bilateral IOP elevation (n = 11, P < 0.001); the ipsilateral IOP increased more noticeably (n = 11, P < 0.05); Bilateral inhibition of PVH projection neurons did not significantly influence IOP (n = 5, P > 0.05). Specific activation of glutamatergic neurons among PVH projection neurons also induced IOP elevation in both eyes (n = 5, P < 0.001). The dorsomedial hypothalamic nucleus, ventromedial hypothalamic nucleus, locus coeruleus and basolateral amygdaloid nucleus responded to light stimulation of PVH in AAV-ChR2 mice. Conclusions: The PVH may play a role in IOP upregulation via glutamatergic neurons.

The efficacy of prevention for colon cancer based on the microbiota therapy and the antitumor mechanisms with intervention of dietary Lactobacillus.

Gut microbiota and their secreted metabolites have an influence on the initiation and progression of colon cancer. Probiotics are extensively perceived as a potential microbiota-modulation strategy to promote the health of the host, while the effectiveness of preventing colon cancer based on microbiota therapy has not been confirmed, and antitumor mechanisms influenced by microbiota and their metabolites with the intervention of probiotics remain to be further investigated. In vitro, Lactobacillus (JY300-8 and JMR-01) significantly inhibited the proliferation of CT26, HT29, and HCT116 cells. Moreover, we studied the prevention and therapy efficiency of Lactobacillus and its underlying antitumor mechanism through the alteration of gut microbiota and their metabolites regulated by Lactobacillus in colon cancer models in mice. We demonstrated that the pre-administration of Lactobacillus (JY300-8 and JMR-01) for 20 days before establishing tumor models resulted in an 86.21% reduction in tumor formation rate compared to tumor control group. Subsequently, continuous oral administration of living Lactobacillus significantly suppresses tumor growth, and tumor volumes decrease by 65.2%. Microbiome and metabolome analyses reveal that Lactobacillus suppresses colonic tumorigenesis and progression through the modulation of gut microbiota homeostasis and metabolites, including the down-regulation of secondary bile acids, sphingosine 1-phosphate (S1P), and pyrimidine metabolism, as well as the production of anticarcinogenic compounds in tumor-bearing mice. Additionally, metabolome analyses of Lactobacillus (JY300-8 and JMR-01) indicate that living Lactobacillus could reduce the relative abundance of alanine and L-serine to suppress tumor progression by regulating the tumor microenvironment, including down-regulation of pyrimidine metabolism and S1P signaling in cancer. These findings provide a potential prevention strategy and therapeutic target for colon cancer through the intervention of dietary Lactobacillus. IMPORTANCE The modulation of gut microbiota and metabolites has a significant influence on the progression of colon cancer. Our research indicated that the intervention of probiotics is a potentially feasible strategy for preventing colon cancer. We have also revealed the underlying antitumor mechanism through the alteration of gut microbiota and their metabolites, which could lead to broader biomedical impacts on the prevention and therapy of colon cancer with microbiota-based therapy regulated by probiotics.

The G285S mutation in nsP1 is sufficient to render Sindbis virus as a stable vector for gene delivery.

Neuroscience, gene therapy, and vaccine have all benefited from the increased use of viral vectors. Sindbis virus (SINV) is a notable candidate among these vectors. However, viral vectors commonly suffer from a loss of expression of the transgene, especially RNA viral vectors. In this study, we used a directed evolution approach by continuous passage of selection to identify adaptive mutations that help SINV to stably express exogenous genes. As a result, we found two adaptive mutations that are located at aa 285 (G to S) of nsP1 and aa 422 (D to G) of nsP2, respectively. Further study showed that G285S was sufficient for SINV to stabilize the expression of the inserted gene, while D422G was not. Combined with AlphaFold2 and sequence alignment with the genus Alphavirus, we found that G285S is conserved. Based on this mutation, we constructed a new vector for the applications in neural circuits mapping. Our results indicated that the mutant SINV maintained its anterograde transsynaptic transmission property. In addition, when the transgene was replaced by another gene, granulocyte-macrophage colony-stimulating factor (GM-CSF), the vector still showed stable expression of the inserted gene. Hence, using SINV as an example, we have demonstrated an efficient approach to greatly augment the gene delivery capacity of viral vectors, which will be useful to neuroscience and oncolytic therapy.

Clinical Efficacy and Safety of AdV-tk Gene Therapy for Patients with Cervical Squamous Intraepithelial Lesion: A Prospective Study.

Cervical cancer is the fourth most common type of cancer for women in 2020, and many more women have cervical precancerous lesion-squamous intraepithelial lesion (SIL). Early treatment of cervical SIL to reverse or delay its progression is an important approach to reduce the incidence of cervical cancer. The efficacy and safety of adenovirus-based vectors expressing the thymidine kinase gene (AdV-tk) in the treatment of multiple types of cancers shows promise for its use in gynecology. We aim to provide relevant clinical efficacy and safety data after introducing AdV-tk for the treatment of cervical SIL for the first time through this prospective study. We conducted a maximum of 6 sessions to administer AdV-tk gene therapy to 23 patients (mean age: 35 years old) with cervical low-/high-grade SIL (LSIL/HSIL) who were enrolled from August 2015 to April 2018 and analyzed the clinical characteristics and follow-up outcomes (mean follow-up period: 7.3 months). The present study consisted of 17 patients (73.9%) with cervical HSIL and 6 patients with LSIL confirmed by colposcopy-directed biopsy. We observed an overall histological remission and regression rate of 87.0% (20/23, 95% confidence interval [95% CI]: 73.2-100, p < 0.001) after AdV-tk gene therapy. Eight patients (34.8%) were detected with human papillomavirus (HPV) 16/18 subtypes and 13 patients were found to be positive with at least one of the other 13 high-risk HPV (HR-HPV) subtypes, while 2 patients did not have any of the 15 HR-HPV subtypes. The overall clearance rate of HR-HPV was 76.2% (16/21, 95% CI: 58.0-94.4, p = 0.016) after AdV-tk gene therapy. For safety evaluation, no severe complications were reported in any of the 23 patients. The most commonly reported symptom was fever in 52.2% (12/23) of patients and all symptoms were fully resolved after symptomatic treatment. Our data indicate that AdV-tk gene therapy has high efficacy and safety in the treatment of cervical SIL among gynecological patients. Our findings provide clinical evidence on the potential promotion and application of AdV-tk in the treatment of cervical SIL, and potentially for cervical cancer, among gynecological patients.

Effects of propofol and sevoflurane on social and anxiety-related behaviours in sleep-deprived rats.

BACKGROUND: Sleep disorders can profoundly affect neurological function. We investigated changes in social and anxiety-related brain functional connectivity induced by sleep deprivation, and the potential therapeutic effects of the general anaesthetics propofol and sevoflurane in rats. METHODS: Twelve-week-old male Sprague-Dawley rats were subjected to sleep deprivation for 20 h per day (from 14:00 to 10:00 the next day) for 4 consecutive weeks. They were free from sleep deprivation for the remaining 4 h during which they received propofol (40 mg kg-1 i.p.) or sevoflurane (2% for 2 h) per day or no treatment. These cohorts were instrumented for EEG/EMG recordings on days 2, 14, and 28. Different cohorts were used for open field and three-chambered social behavioural tests, functional MRI, nuclear magnetic resonance spectroscopy, and positron emission tomography imaging 48 h after 4 weeks of sleep deprivation. RESULTS: Propofol protected against sleep deprivation-induced anxiety behaviours with more time (44.7 [8.9] s vs 24.2 [4.1] s for the sleep-deprivation controls; P<0.001) spent in the central area of the open field test and improved social preference index by 30% (all P<0.01). Compared with the sleep-deprived rats, propofol treatment enhanced overall functional connectivity by 74% (P<0.05) and overall glucose metabolism by 30% (P<0.01), and improved glutamate kinetics by 20% (P<0.05). In contrast, these effects were not found after sevoflurane treatment. CONCLUSIONS: Unlike sevoflurane, propofol reduced sleep deprivation-induced social and anxiety-related behaviours. Propofol might be superior to sevoflurane for patients with sleep disorders who receive anaesthesia, which should be studied in clinical studies.

Investigations of brain-wide functional and structural networks of dopaminergic and CamKIIα-positive neurons in VTA with DREADD-fMRI and neurotropic virus tracing technologies.

BACKGROUND: The ventral tegmental area (VTA) contains heterogeneous cell populations. The dopaminergic neurons in VTA play a central role in reward and cognition, while CamKIIα-positive neurons, composed mainly of glutamatergic and some dopaminergic neurons, participate in the reward learning and locomotor activity behaviors. The differences in brain-wide functional and structural networks between these two neuronal subtypes were comparatively elucidated. METHODS: In this study, we applied a method combining Designer Receptors Exclusively Activated by Designer Drugs (DREADD) and fMRI to assess the cell type-specific modulation of whole-brain neural networks. rAAV encoding the cre-dependent hM3D was injected into the right VTA of DAT-cre or CamKIIα-cre transgenic rats. The global brain activities elicited by DREADD stimulation were then detected using BOLD-fMRI. Furthermore, the cre-dependent antegrade transsynaptic viral tracer H129ΔTK-TT was applied to label the outputs of VTA neurons. RESULTS: We found that DREADD stimulation of dopaminergic neurons induced significant BOLD signal changes in the VTA and several VTA-related regions including mPFC, Cg and Septum. More regions responded to selective activation of VTA CamKIIα-positive neurons, resulting in increased BOLD signals in VTA, Insula, mPFC, MC_R (Right), Cg, Septum, Hipp, TH_R, PtA_R, and ViC_R. Along with DREADD-BOLD analysis, further neuronal tracing identified multiple cortical (MC, mPFC) and subcortical (Hipp, TH) brain regions that are structurally and functionally connected by VTA dopaminergic and CamKIIα-positive neurons. CONCLUSIONS: Our study dissects brain-wide structural and functional networks of two neuronal subtypes in VTA and advances our understanding of VTA functions.

Overexpression of Sirt6 ameliorates sleep deprivation induced-cognitive impairment by modulating glutamatergic neuron function.

Sleep benefits the restoration of energy metabolism and thereby supports neuronal plasticity and cognitive behaviors. Sirt6 is a NAD+-dependent protein deacetylase that has been recognized as an essential regulator of energy metabolism because it modulates various transcriptional regulators and metabolic enzymes. The aim of this study was to investigate the influence of Sirt6 on cerebral function after chronic sleep deprivation (CSD). We assigned C57BL/6J mice to control or two CSD groups and subjected them to AAV2/9-CMV-EGFP or AAV2/9-CMV-Sirt6-EGFP infection in the prelimbic cortex (PrL). We then assessed cerebral functional connectivity (FC) using resting-state functional MRI, neuron/astrocyte metabolism using a metabolic kinetics analysis; dendritic spine densities using sparse-labeling; and miniature excitatory postsynaptic currents (mEPSCs) and action potential (AP) firing rates using whole-cell patch-clamp recordings. In addition, we evaluated cognition via a comprehensive set of behavioral tests. Compared with controls, Sirt6 was significantly decreased (P < 0.05) in the PrL after CSD, accompanied by cognitive deficits and decreased FC between the PrL and accumbens nucleus, piriform cortex, motor cortex, somatosensory cortex, olfactory tubercle, insular cortex, and cerebellum. Sirt6 overexpression reversed CSD-induced cognitive impairment and reduced FC. Our analysis of metabolic kinetics using [1-13C] glucose and [2-13C] acetate showed that CSD reduced neuronal Glu4 and GABA2 synthesis, which could be fully restored via forced Sirt6 expression. Furthermore, Sirt6 overexpression reversed CSD-induced decreases in AP firing rates as well as the frequency and amplitude of mEPSCs in PrL pyramidal neurons. These data indicate that Sirt6 can improve cognitive impairment after CSD by regulating the PrL-associated FC network, neuronal glucose metabolism, and glutamatergic neurotransmission. Thus, Sirt6 activation may have potential as a novel strategy for treating sleep disorder-related diseases.

AAV11 enables efficient retrograde targeting of projection neurons and enhances astrocyte-directed transduction.

Viral tracers that enable efficient retrograde labeling of projection neurons are powerful vehicles for structural and functional dissections of the neural circuit and for the treatment of brain diseases. Currently, some recombinant adeno-associated viruses (rAAVs) based on capsid engineering are widely used for retrograde tracing, but display undesirable brain area selectivity due to inefficient retrograde transduction in certain neural connections. Here we developed an easily editable toolkit to produce high titer AAV11 and demonstrated that it exhibits potent and stringent retrograde labeling of projection neurons in adult male wild-type or Cre transgenic mice. AAV11 can function as a powerful retrograde viral tracer complementary to AAV2-retro in multiple neural connections. In combination with fiber photometry, AAV11 can be used to monitor neuronal activities in the functional network by retrograde delivering calcium-sensitive indicator under the control of a neuron-specific promoter or the Cre-lox system. Furthermore, we showed that GfaABC1D promoter embedding AAV11 is superior to AAV8 and AAV5 in astrocytic tropism in vivo, combined with bidirectional multi-vector axoastrocytic labeling, AAV11 can be used to study neuron-astrocyte connection. Finally, we showed that AAV11 allows for analyzing circuit connectivity difference in the brains of the Alzheimer's disease and control mice. These properties make AAV11 a promising tool for mapping and manipulating neural circuits and for gene therapy of some neurological and neurodegenerative disorders.

CLEAR Strategy Inhibited HSV Proliferation Using Viral Vectors Delivered CRISPR-Cas9.

Herpes simplex virus type 1 (HSV-1) is a leading cause of encephalitis and infectious blindness. The commonly used clinical therapeutic drugs are nucleoside analogues such as acyclovir. However, current drugs for HSV cannot eliminate the latent virus or viral reactivation. Therefore, the development of new treatment strategies against latent HSV has become an urgent need. To comprehensively suppress the proliferation of HSV, we designed the CLEAR strategy (coordinated lifecycle elimination against viral replication). VP16, ICP27, ICP4, and gD-which are crucial genes that perform significant functions in different stages of the HSV infection lifecycle-were selected as targeting sites based on CRISPR-Cas9 editing system. In vitro and in vivo investigations revealed that genome editing by VP16, ICP27, ICP4 or gD single gene targeting could effectively inhibit HSV replication. Moreover, the combined administration method (termed "Cocktail") showed superior effects compared to single gene editing, which resulted in the greatest decrease in viral proliferation. Lentivirus-delivered CRISPR-Cas9/gRNA editing could effectively block HSV replication. The CLEAR strategy may provide new insights into the potential treatment of refractory HSV-1-associated diseases, particularly when conventional approaches have encountered resistance.

A direct spino-cortical circuit bypassing the thalamus modulates nociception.

Nociceptive signals are usually transmitted to layer 4 neurons in somatosensory cortex via the spinothalamic-thalamocortical pathway. The layer 5 corticospinal neurons in sensorimotor cortex are reported to receive the output of neurons in superficial layers; and their descending axons innervate the spinal cord to regulate basic sensorimotor functions. Here, we show that a subset of layer 5 neurons receives spinal inputs through a direct spino-cortical circuit bypassing the thalamus, and thus define these neurons as spino-cortical recipient neurons (SCRNs). Morphological studies revealed that the branches from spinal ascending axons formed a kind of disciform structure with the descending axons from SCRNs in the basilar pontine nucleus (BPN). Electron microscopy and calcium imaging further confirmed that the axon terminals from spinal ascending neurons and SCRNs made functional synaptic contacts in the BPN, linking the ascending sensory pathway to the descending motor control pathway. Furthermore, behavioral tests indicated that the spino-cortical connection in the BPN was involved in nociceptive responses. In vivo calcium imaging showed that SCRNs responded to peripheral noxious stimuli faster than neighboring layer 4 cortical neurons in awake mice. Manipulating activities of SCRNs could modulate nociceptive behaviors. Therefore, this direct spino-cortical circuit represents a noncanonical pathway, allowing a fast sensory-motor transition of the brain in response to noxious stimuli.

Astrocytic α4-containing nAChR signaling in the hippocampus governs the formation of temporal association memory.

Everyday episodic memories involve linking together related events that are temporally separated. However, the mechanisms of forming this temporal association have remained unclear. Here, using astrocyte-specific manipulations, we show that potentiating astrocyte Ca2+ signaling in the hippocampal cornu ammonis 1 (CA1) enhances the strength of such temporal association, in parallel with long-term potentiation (LTP) enhancement of temporoammonic pathway to CA1, whereas attenuation of astrocyte Ca2+ signaling has the opposite effect. Moreover, we identify that these effects are mediated by astrocytic α4 subunit-containing nicotinic acetylcholine receptors (α4-nAChRs) via mechanisms involving NMDAR co-agonist supply. Finally, astrocytic α4-nAChRs underlie the cognitive enhancer nicotine's physiological effects. Together, these findings highlight the importance of astrocyte Ca2+ signaling in cognitive behavior and reveal a mechanism in governing the temporal association of episodic memory formation that operates through α4-nAChRs on hippocampal astrocytes.