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1.
Elife ; 132024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38390967

RESUMO

Deciphering patterns of connectivity between neurons in the brain is a critical step toward understanding brain function. Imaging-based neuroanatomical tracing identifies area-to-area or sparse neuron-to-neuron connectivity patterns, but with limited throughput. Barcode-based connectomics maps large numbers of single-neuron projections, but remains a challenge for jointly analyzing single-cell transcriptomics. Here, we established a rAAV2-retro barcode-based multiplexed tracing method that simultaneously characterizes the projectome and transcriptome at the single neuron level. We uncovered dedicated and collateral projection patterns of ventromedial prefrontal cortex (vmPFC) neurons to five downstream targets and found that projection-defined vmPFC neurons are molecularly heterogeneous. We identified transcriptional signatures of projection-specific vmPFC neurons, and verified Pou3f1 as a marker gene enriched in neurons projecting to the lateral hypothalamus, denoting a distinct subset with collateral projections to both dorsomedial striatum and lateral hypothalamus. In summary, we have developed a new multiplexed technique whose paired connectome and gene expression data can help reveal organizational principles that form neural circuits and process information.


Assuntos
Neuritos , Neurônios , Neurônios/metabolismo , Encéfalo , Córtex Pré-Frontal , Vias Neurais/fisiologia
2.
Science ; 383(6682): eadj9198, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38300992

RESUMO

Mapping single-neuron projections is essential for understanding brain-wide connectivity and diverse functions of the hippocampus (HIP). Here, we reconstructed 10,100 single-neuron projectomes of mouse HIP and classified 43 projectome subtypes with distinct projection patterns. The number of projection targets and axon-tip distribution depended on the soma location along HIP longitudinal and transverse axes. Many projectome subtypes were enriched in specific HIP subdomains defined by spatial transcriptomic profiles. Furthermore, we delineated comprehensive wiring diagrams for HIP neurons projecting exclusively within the HIP formation (HPF) and for those projecting to both intra- and extra-HPF targets. Bihemispheric projecting neurons generally projected to one pair of homologous targets with ipsilateral preference. These organization principles of single-neuron projectomes provide a structural basis for understanding the function of HIP neurons.


Assuntos
Axônios , Mapeamento Encefálico , Hipocampo , Neurônios , Animais , Camundongos , Axônios/fisiologia , Axônios/ultraestrutura , Hipocampo/ultraestrutura , Neurônios/classificação , Neurônios/ultraestrutura , Análise de Célula Única/métodos , Rede Nervosa , Masculino , Camundongos Endogâmicos C57BL
3.
Cell Rep ; 43(2): 113756, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38358886

RESUMO

Short-term memory (STM) maintains information during a short delay period. How long-range and local connections interact to support STM encoding remains elusive. Here, we tackle the problem focusing on long-range projections from the medial prefrontal cortex (mPFC) to the anterior agranular insular cortex (aAIC) in head-fixed mice performing an olfactory delayed-response task. Optogenetic and electrophysiological experiments reveal the behavioral importance of the two regions in encoding STM information. Spike-correlogram analysis reveals strong local and cross-region functional coupling (FC) between memory neurons encoding the same information. Optogenetic suppression of mPFC-aAIC projections during the delay period reduces behavioral performance, the proportion of memory neurons, and memory-specific FC within the aAIC, whereas optogenetic excitation enhances all of them. mPFC-aAIC projections also bidirectionally modulate the efficacy of STM-information transfer, measured by the contribution of FC spiking pairs to the memory-coding ability of following neurons. Thus, prefrontal projections modulate insular neurons' functional connectivity and memory-coding ability to support STM.


Assuntos
Córtex Insular , Memória de Curto Prazo , Animais , Camundongos , Citoplasma , Neurônios , Optogenética
4.
Nat Neurosci ; 25(4): 515-529, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35361973

RESUMO

Prefrontal cortex (PFC) is the cognitive center that integrates and regulates global brain activity. However, the whole-brain organization of PFC axon projections remains poorly understood. Using single-neuron reconstruction of 6,357 mouse PFC projection neurons, we identified 64 projectome-defined subtypes. Each of four previously known major cortico-cortical subnetworks was targeted by a distinct group of PFC subtypes defined by their first-order axon collaterals. Further analysis unraveled topographic rules of soma distribution within PFC, first-order collateral branch point-dependent target selection and terminal arbor distribution-dependent target subdivision. Furthermore, we obtained a high-precision hierarchical map within PFC and three distinct functionally related PFC modules, each enriched with internal recurrent connectivity. Finally, we showed that each transcriptome subtype corresponds to multiple projectome subtypes found in different PFC subregions. Thus, whole-brain single-neuron projectome analysis reveals organization principles of axon projections within and outside PFC and provides the essential basis for elucidating neuronal connectivity underlying diverse PFC functions.


Assuntos
Neurônios , Córtex Pré-Frontal , Animais , Axônios , Encéfalo , Interneurônios , Camundongos , Neurônios/fisiologia , Córtex Pré-Frontal/fisiologia
5.
Neuron ; 105(5): 934-946.e5, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32135091

RESUMO

Whether transient or sustained neuronal activity during the delay period underlies working memory (WM) has been debated. Here, we report that transient, but not sustained, delay-period activity in mouse anterior agranular insular cortex (aAIC) plays a dominant role in maintaining WM information during learning of novel olfactory tasks. By optogenetic screening over 12 brain regions, we found that suppressing aAIC activity markedly impaired olfactory WM maintenance during learning. Single-unit recording showed that odor-selective aAIC neurons with predominantly transient firing patterns encoded WM information. Both WM task performance and transient-neuron proportion were enhanced and reduced by activating and suppressing the delay-period activity of the projection from medial prefrontal cortex (mPFC) to aAIC. The ability of mice to resist delay-period distractors also correlated with an increased percentage of transient neurons. Therefore, transient, but not sustained, aAIC neuronal activity during the delay period is largely responsible for maintaining information while learning novel WM tasks.


Assuntos
Córtex Cerebral/metabolismo , Aprendizagem/fisiologia , Memória de Curto Prazo/fisiologia , Neurônios/metabolismo , Córtex Pré-Frontal/metabolismo , Olfato , Animais , Atenção/fisiologia , Córtex Cerebral/citologia , Camundongos , Vias Neurais , Optogenética
6.
Elife ; 82019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31232695

RESUMO

Working memory is a critical brain function for maintaining and manipulating information over delay periods of seconds. It is debated whether delay-period neural activity in sensory regions is important for the active maintenance of information during the delay period. Here, we tackle this question by examining the anterior piriform cortex (APC), an olfactory sensory cortex, in head-fixed mice performing several olfactory working memory tasks. Active information maintenance is necessary in these tasks, especially in a dual-task paradigm in which mice are required to perform another distracting task while actively maintaining information during the delay period. Optogenetic suppression of neuronal activity in APC during the delay period impaired performance in all the tasks. Furthermore, electrophysiological recordings revealed that APC neuronal populations encoded odor information in the delay period even with an intervening distracting task. Thus, delay activity in APC is important for active information maintenance in olfactory working memory.


Assuntos
Memória de Curto Prazo , Neurônios/fisiologia , Córtex Piriforme/fisiologia , Animais , Eletroencefalografia , Camundongos , Optogenética
7.
Science ; 346(6208): 458-63, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25342800

RESUMO

Cognitive processes require working memory (WM) that involves a brief period of memory retention known as the delay period. Elevated delay-period activity in the medial prefrontal cortex (mPFC) has been observed, but its functional role in WM tasks remains unclear. We optogenetically suppressed or enhanced activity of pyramidal neurons in mouse mPFC during the delay period. Behavioral performance was impaired during the learning phase but not after the mice were well trained. Delay-period mPFC activity appeared to be more important in memory retention than in inhibitory control, decision-making, or motor selection. Furthermore, endogenous delay-period mPFC activity showed more prominent modulation that correlated with memory retention and behavioral performance. Thus, properly regulated mPFC delay-period activity is critical for information retention during learning of a WM task.


Assuntos
Aprendizagem/fisiologia , Memória de Curto Prazo/fisiologia , Córtex Pré-Frontal/fisiologia , Células Piramidais/fisiologia , Retenção Psicológica , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Channelrhodopsins , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Córtex Pré-Frontal/citologia , Tempo de Reação , Olfato
8.
CNS Neurosci Ther ; 20(11): 951-60, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25042179

RESUMO

AIMS: Our previous studies indicate that glycine can inhibit N-methyl-D-aspartate receptor (NMDAR) responses induced by high concentrations of NMDA in rat hippocampal neurons. The present study was designed to observe whether D-serine induces inactivation of NMDARs in cultured rat hippocampal neurons and to investigate the underlying mechanisms of this effect. METHODS: Cell culture, whole-cell patch-clamp electrophysiology, Ca(2+) imaging, immunohistochemistry, and Western blot analysis were used. RESULTS: We found that the peak current and Ca(2+) influx evoked by 30 µM NMDA were increased by co-application of D-serine, but those evoked by 300 µM NMDA were reduced dose-dependently by co-application of D-serine. However, the inhibitory effect of D-serine on NMDAR responses was reversed by ZnCl2 (30 nM), an inhibitor of the NR2A subunit, but was less influenced by ifenprodil (10 µM), an NR2B inhibitor. In addition, the inhibitory effect of D-serine was not detected in young hippocampal neurons that expressed less of the NR2A subunits and reversed in the presence of 10 mM BAPTA. CONCLUSIONS: D-serine can also induce inactivation of NMDARs, the NR2A subunit is required for the induction of this effect, and this inactivation is Ca(2+)-dependent in nature. This action of D-serine is hypothesized to play a neuroprotective role upon a sustained large glutamate insult to the brain.


Assuntos
Cálcio/metabolismo , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Embrião de Mamíferos , Agonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/citologia , Potenciais da Membrana/efeitos dos fármacos , N-Metilaspartato/farmacologia , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Ratos , Ratos Sprague-Dawley
9.
Sheng Li Xue Bao ; 65(2): 135-42, 2013 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-23598868

RESUMO

Migration of vascular smooth muscle cells (VSMCs) is involved in vascular development and various vascular diseases; however, the molecular mechanisms of VSMC migration remain unclear. In this study, we established an inverted coverslip migration assay to study the migratory properties of cultured VSMCs on extracellular matrix. Pulmonary arterial smooth muscle cells (PASMCs) from rats were cultured and identified by immunocytochemistry. Each coverslip with a confluent monolayer of PASMCs was inverted to a larger coverslip which was coated with phosphate buffered saline (PBS, as a control), poly-D-lysine hydrobromide (PDL), laminin or Matrigel. After 24 h of migration over the larger coverslip, PASMCs were fixed, and reliably quantified. The roles and mechanisms of extracellular matrix in PASMC migration were further studied by wound-healing assay and immunocytochemistry. The results showed that: (1) The purity of the cultured PASMCs was (97 ± 3)%. (2) The number of PASMCs on laminin or Matrigel migrating out from the inverted coverslip was significantly increased compared with that on PBS or PDL, and the migratory distance of PASMCs on laminin or Matrigel was significantly farther than that on PBS or PDL. (3) The motility of PASMCs on laminin or Matrigel was significantly higher than that on PBS at 8 h, 12 h and 24 h after wounding, respectively. (4) F-actin staining showed that F-actin was congregated along the brim of the migrating cells from the inverted coverslip, and vinculin (a cell marker of focal adhesion) staining showed that the distribution of vinculin in PASMCs plated on laminin or Matrigel was significantly lower than that on PBS or PDL. These results suggest that the inverted coverslip migration assay is suitable to study VSMC migration, and laminin and Matrigel substrates may promote VSMC migration through inhibiting the formation of focal adhesion and regulating the cytoskeletal proteins.


Assuntos
Movimento Celular , Matriz Extracelular/química , Miócitos de Músculo Liso/citologia , Actinas/química , Animais , Adesão Celular , Células Cultivadas , Colágeno/química , Combinação de Medicamentos , Laminina/química , Músculo Liso Vascular/citologia , Proteoglicanas/química , Artéria Pulmonar/citologia , Ratos
10.
Sci China Life Sci ; 55(12): 1075-81, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23233222

RESUMO

In the presence of glutamate and co-agonists, e.g., glycine, the N-methyl-D-aspartate receptor (NMDAR) plays an important role in physiological and pathophysiological brain processes. Previous studies indicate glycine could inhibit NMDAR responses induced by high concentration of NMDA in hippocampal neurons. The mechanism underlying this inhibitory impact, however, has been unclear. In this study, the whole-cell patch-clamp recording and Ca(2+) imaging with Fluo-3/AM under laser scanning confocal microscope were used to analyze the possible involvement of NMDAR subunits in this effect. We found that the peak current of NMDARs and Ca(2+) influx induced by high concentration of NMDA were reduced by treatment of glycine (0.03-10 µmol L(-1)) in a dose-dependent manner, and that the glycine-dependent inhibition of NMDAR responses, which were induced at 300 µmol L(-1) NMDA, was reversed by ZnCl(2) through the blocking of the NR2A subunit of NMDARs, but was less influenced by ifenprodil, a NR2B inhibitor. Our results suggest that the glycine-dependent inactivation of NMDARs is potentially modulated by the regulatory subunit NR2A.


Assuntos
Glicina/farmacologia , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Zinco/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Hipocampo/citologia , Hipocampo/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley
11.
Artigo em Chinês | MEDLINE | ID: mdl-22097712

RESUMO

OBJECTIVE: To investigate the changes of endoplasmic reticulum stress-induced apoptosis in pulmonary tissue of rats with hypoxic pulmonary hypertension. METHODS: Twenty two male SD rats were randomly divided into control group and 4-week hypoxia-hypercapnia group (n=11). The mean pulmonary arterial pressure (mPAP) and the mean carotid arterial pressure (mCAP) were monitored, and the weight ratio of right ventricle (RV) to left ventricle plus septum (LV + S) were measured. The rattish pathological model were assessed by mPAP, mCAP, RV/(LV+ S), vessel wall area/total area (WA/TA), vessel cavity area/total area (CA/TA) and media thickness of pulmonary arteriole (PAMT). The pulmonary apoptotic cells were detected by Hoechst staining. RT-PCR was used to study the genetic expression of caspasel2, glucose regulated protein 78 (GRP78) and GRP94 in pulmonary tissue. The expression of GRP94 and GRP78 proteins in pulmonary tissue were determined by using immunohistochemistry. RESULTS: (1) (The mPAP, RV/(LV + S), WA/TA and PAMT were respectively higher by 50.5%, 37.3%, 72.5% and 137% in hypoxic group than those in control group, while CA/TA was lower by 41.9% (all P < 0.01). There was not significant difference of mCAP between the two groups. (2) Hoechst staining showed that the pulmonary apoptotic cells in hypoxic group outnumbered markedly than those in control group, and the apoptotic cells were mainly in pulmonary tissue, while they were rare in pulmonary vascular smooth muscle cell. (3) Compared with control group, the expression of pulmonary caspasel2, GRP78 and GRP94 mRNA in hypoxic group were higher by 144%, 137% and 80.7% (all P < 0.05), respectively. (4) The expression of pulmonary GRP78 and GRP94 proteins were up-regulated in hypoxic group, and these proteins mainly localized in pulmonary vascular endothelial cell. CONCLUSION: The endoplasmic reticulum stress-induced apoptosis may be one of the mechanism of hypoxic pulmonary hypertension and pulmonary vascular wall remodeling.


Assuntos
Apoptose/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Hipertensão Pulmonar/fisiopatologia , Hipóxia/fisiopatologia , Pulmão/patologia , Animais , Caspase 12/metabolismo , Proteínas de Choque Térmico/metabolismo , Hipercapnia/fisiopatologia , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Hipóxia/complicações , Masculino , Glicoproteínas de Membrana/metabolismo , Ratos , Ratos Sprague-Dawley
12.
Sheng Li Xue Bao ; 63(3): 238-44, 2011 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-21681342

RESUMO

Oxidative stress could induce apoptosis and autophagy process simultaneously, but the role of autophagy is still not clear. Beclin 1, a key gene regulating the preautophagosome formation, is involved in the injury induced by oxidative stress. To observe the role of autophagy in H2O2-induced injury of U251 cells, the recombinant plasmid Psilencer3.1-siRNA-Beclin 1 was transfected into U251 cells by eukaryotic cell transfection technique. Plasmid vector and cell culture medium were used as negative and control groups respectively. The cells were collected 24 h later, and the cell total protein was extracted to detect Beclin 1, Bcl-2 and Bax protein expressions by Western blot. After the Beclin 1-siRNA cells were treated with 1 mmol/L H2O2, the autophagic vacuoles in the cells were stained with monodansylcadaverine (MDC), and the cell apoptotic ratio was determined with PI/Annexin V-FITC staining by flow cytometry analysis. The results showed that the synthetic siRNA decreased the expression of Beclin 1 protein significantly, but had no obvious effect on the levels of Bcl-2 and Bax protein expressions. Compared with those in the control group, the autophagic vacuoles, the level of LC3-II protein expression and the percentage of apoptotic cells increased (P < 0.05) in 1 mmol/L H2O2 group. In Beclin 1-siRNA + H2O2 group, autophagic vacuoles and the levels of LC3-II protein expression decreased obviously, the percentage of apoptotic cells increased significantly compared with that in 1 mmol/L H2O2 group (P < 0.05). H2O2 and autophagy inhibitor 3-methyladenine (3-MA) combination also increased the percentage of apoptotic cells obviously (P < 0.05). These results revealed that the transfection of Psilencer3.1-siRNA-Beclin 1 effectively inhibited the expression of Beclin 1 protein expression, degraded the autophagy level and increased the apoptotic rate in U251 cells under oxidative stress, which was coincident with the effect of autophagy inhibitor 3-MA. This study suggests that autophagy is a cell protective role in oxidative stress process, and the inhibition of autophagy may enhance apoptosis.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/fisiologia , Glioma/patologia , Peróxido de Hidrogênio/farmacologia , Proteínas de Membrana/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteína Beclina-1 , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/genética , Estresse Oxidativo , RNA Interferente Pequeno/genética , Transfecção
13.
J Neurosci Res ; 88(9): 2035-45, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20186763

RESUMO

To investigate the neuroprotective effect of L-serine and its underlying mechanisms, focal cerebral ischemia was induced in rats by occlusion of middle cerebral artery (MCAO) with a suture, and reperfusion was given by filament withdrawal 2 hr later. Meanwhile, rat hippocampal neurons were primarily cultured, and incubated in serum-free medium in an incubator containing 1% O(2) for hypoxic exposure of 5 hr, or incubated in serum-free medium containing 1 mM glutamate for glutamate exposure of 2 hr. Brain tissue injury and cell damage were then measured. L-serine dose-dependently decreased the neurology deficit score and infarct volume, elevated the cell viability and inhibited the leakage of lactate dehydrogenase. These effects were blocked by strychnine in both MCAO rats and cultured hippocampal neurons. Furthermore, L-serine (168 mg.kg(-1)) reduced the brain water content, permeability of blood-brain barrier, neuronal loss and the expression of activated caspase-3 in the cortex. In addition, L-serine effectively protected the brain from damage when it was administered within 6 hr after the end of MCAO. It is suggested that L-serine could exert a neuroprotective effect on the ischemic-reperfused brain and on the hypoxia- or glutamate-exposed hippocampal neurons, which may be mediated by activating glycine receptors.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Serina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/fisiopatologia , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Ácido Glutâmico/metabolismo , Glicinérgicos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/fisiopatologia , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/fisiologia , Fármacos Neuroprotetores/administração & dosagem , Ratos , Ratos Sprague-Dawley , Serina/administração & dosagem , Estricnina/farmacologia , Fatores de Tempo
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