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The lateral septum (LS) is composed of heterogeneous cell types that are important for various motivated behaviors. However, the transcriptional profiles, spatial arrangement, function, and connectivity of these cell types have not been systematically studied. Using single-nucleus RNA sequencing, we delineated diverse genetically defined cell types in the LS that play distinct roles in reward processing. Notably, we found that estrogen receptor 1 (Esr1)-expressing neurons in the ventral LS (LSEsr1) are key drivers of reward seeking via projections to the ventral tegmental area, and these neurons play an essential role in methamphetamine (METH) reward and METH-seeking behavior. Extended exposure to METH increases the excitability of LSEsr1 neurons by upregulating hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, thereby contributing to METH-induced locomotor sensitization. These insights not only elucidate the intricate molecular, circuit, and functional architecture of the septal region in reward processing but also reveal a neural pathway critical for METH reward and behavioral sensitization.
Assuntos
Metanfetamina , Neurônios , Recompensa , Núcleos Septais , Animais , Camundongos , Neurônios/fisiologia , Neurônios/metabolismo , Metanfetamina/farmacologia , Núcleos Septais/fisiologia , Núcleos Septais/metabolismo , Masculino , Área Tegmentar Ventral/fisiologia , Área Tegmentar Ventral/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Vias Neurais/fisiologia , Camundongos Endogâmicos C57BL , Comportamento de Procura de Droga/fisiologiaRESUMO
Quantitative analysis of activated neurons in mouse brains by a specific stimulation is usually a primary step to locate the responsive neurons throughout the brain. However, it is challenging to comprehensively and consistently analyze the neuronal activity trace in whole brains of a large cohort of mice from many terabytes of volumetric imaging data. Here, we introduce NEATmap, a deep learning-based high-efficiency, high-precision and user-friendly software for whole-brain neuronal activity trace mapping by automated segmentation and quantitative analysis of immunofluorescence labeled c-Fos+ neurons. We applied NEATmap to study the brain-wide differentiated neuronal activation in response to physical and psychological stressors in cohorts of mice.
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Synapses at dendritic branches exhibit specific properties for information processing. However, how the synapses are orchestrated to dynamically modify their properties, thus optimizing information processing, remains elusive. Here, we observed at hippocampal dendritic branches diverse configurations of synaptic connectivity, two extremes of which are characterized by low transmission efficiency, high plasticity and coding capacity, or inversely. The former favors information encoding, pertinent to learning, while the latter prefers information storage, relevant to memory. Presynaptic intracellular Mg2+ crucially mediates the dynamic transition continuously between the two extreme configurations. Consequently, varying intracellular Mg2+ levels endow individual branches with diverse synaptic computations, thus modulating their ability to process information. Notably, elevating brain Mg2+ levels in aging animals restores synaptic configuration resembling that of young animals, coincident with improved learning and memory. These findings establish intracellular Mg2+ as a crucial factor reconfiguring synaptic connectivity at dendrites, thus optimizing their branch-specific properties in information processing.
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Dendritos , Hipocampo , Magnésio , Plasticidade Neuronal , Sinapses , Transmissão Sináptica , Animais , Magnésio/metabolismo , Sinapses/fisiologia , Sinapses/metabolismo , Hipocampo/fisiologia , Hipocampo/metabolismo , Plasticidade Neuronal/fisiologia , Dendritos/fisiologia , Dendritos/metabolismo , Transmissão Sináptica/fisiologia , Masculino , Memória/fisiologia , Ratos , Aprendizagem/fisiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Amino acid formula (AAF) is increasingly consumed in infants with cow's milk protein allergy; however, the long-term influences on health are less described. In this study, we established a mouse model by subjecting neonatal mice to an amino acid diet (AAD) to mimic the feeding regimen of infants on AAF. Surprisingly, AAD-fed mice exhibited dysbiotic microbiota and increased neuronal activity in both the intestine and brain, as well as gastrointestinal peristalsis disorders and depressive-like behavior. Furthermore, fecal microbiota transplantation from AAD-fed mice or AAF-fed infants to recipient mice led to elevated neuronal activations and exacerbated depressive-like behaviors compared to that from normal chow-fed mice or cow's-milk-formula-fed infants, respectively. Our findings highlight the necessity to avoid the excessive use of AAF, which may influence the neuronal development and mental health of children.
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Microbiota , Hipersensibilidade a Leite , Humanos , Lactente , Feminino , Bovinos , Criança , Animais , Camundongos , Fórmulas Infantis/química , Aminoácidos , DisbioseRESUMO
To investigate the circuit-level neural mechanisms of behavior, simultaneous imaging of neuronal activity in multiple cortical and subcortical regions is highly desired. Miniature head-mounted microscopes offer the capability of calcium imaging in freely behaving animals. However, implanting multiple microscopes on a mouse brain remains challenging due to space constraints and the cumbersome weight of the equipment. Here, we present TINIscope, a Tightly Integrated Neuronal Imaging microscope optimized for electronic and opto-mechanical design. With its compact and lightweight design of 0.43 g, TINIscope enables unprecedented simultaneous imaging of behavior-relevant activity in up to four brain regions in mice. Proof-of-concept experiments with TINIscope recorded over 1000 neurons in four hippocampal subregions and revealed concurrent activity patterns spanning across these regions. Moreover, we explored potential multi-modal experimental designs by integrating additional modules for optogenetics, electrical stimulation or local field potential recordings. Overall, TINIscope represents a timely and indispensable tool for studying the brain-wide interregional coordination that underlies unrestrained behaviors.
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The brain consists of the left and right cerebral hemispheres and both are connected by callosal projections. Less is known about the basic mechanism of this cortical-cortical connection and its functional importance. Here we investigate the cortical-cortical connection between the bilateral anterior cingulate cortex (ACC) by using the classic electrophysiological and optogenetic approach. We find that there is a direct synaptic projection from one side ACC to the contralateral ACC. Glutamate is the major excitatory transmitter for bilateral ACC connection, including projections to pyramidal cells in superficial (II/III) and deep (V/VI) layers of the ACC. Both AMPA and kainate receptors contribute to synaptic transmission. Repetitive stimulation of the projection also evoked postsynaptic Ca2+ influx in contralateral ACC pyramidal neurons. Behaviorally, light activation of the ACC-ACC connection facilitated behavioral withdrawal responses to mechanical stimuli and noxious heat. In an animal model of neuropathic pain, light inhibitory of ACC-ACC connection reduces both primary and secondary hyperalgesia. Our findings provide strong direct evidence for the excitatory or facilitatory contribution of ACC-ACC connection to pain perception, and this mechanism may provide therapeutic targets for future treatment of chronic pain and related emotional disorders.
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Giro do Cíngulo , Neuralgia , Camundongos , Animais , Giro do Cíngulo/fisiologia , Transmissão Sináptica/fisiologia , Células Piramidais , Ácido GlutâmicoRESUMO
Huntington's disease (HD) usually causes cognitive disorders, including learning difficulties, that emerge before motor symptoms. Mutations related to lysosomal trafficking are linked to the pathogenesis of neurological diseases, whereas the cellular mechanisms remain elusive. Here, we discover a reduction in the dendritic density of lysosomes in the hippocampus that correlates with deficits in synaptic plasticity and spatial learning in early CAG-140 HD model mice. We directly manipulate intraneuronal lysosomal positioning with light-induced CRY2:CIB1 dimerization and demonstrate that lysosomal abundance in dendrites positively modulates long-term potentiation of glutamatergic synapses onto the neuron. This modulation depends on lysosomal Ca2+ release, which further promotes endoplasmic reticulum (ER) entry into spines. Importantly, optogenetically restoring lysosomal density in dendrites rescues the synaptic plasticity deficit in hippocampal slices of CAG-140 mice. Our data reveal dendritic lysosomal density as a modulator of synaptic plasticity and suggest a role of lysosomal mispositioning in cognitive decline in HD.
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Doença de Huntington , Camundongos , Animais , Doença de Huntington/genética , Plasticidade Neuronal/fisiologia , Neurônios/patologia , Hipocampo/patologia , Sinapses/patologia , Lisossomos/patologia , Dendritos/patologia , Espinhas Dendríticas/patologiaRESUMO
Peripheral neural interfaces, potent in modulating local and systemic immune responses for disease treatment, face significant challenges due to the peripheral nerves' broad distribution in tissues like the fascia, periosteum, and skin. The incongruity between static electronic components and the dynamic, complex organization of the peripheral nervous system often leads to interface failure, stalling circuit research and clinical applications. To overcome these, we developed a self-assembling, tissue-adaptive electrode composed of a single-component cocktail nanosheet colloid, including dopants, conducting polymers, stabilizers, and an MXene catalyst. Delivered via a jet injector to designated nerve terminals, this assembly utilizes reactive oxygen species to catalytically dope poly (3,4-ethylenedioxythiophene), enhancing π-π interactions between nanosheets, and yielding a conductive, biodegradable interface. This interface effectively regulates local immune activity and promotes sensory and motor nerve functional restoration in nerve-injured mice, while engaging the vagal-adrenal axis in freely moving mice, eliciting catecholamine neurotransmitter release, and suppressing systemic cytokine storms. This innovative strategy specifically targets nerve substructures, bolstering local and systemic immune modulation, and paving the way for the development of self-adaptive dynamic neural interfaces.
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Nervos Periféricos , Sistema Nervoso Periférico , Camundongos , Animais , Polímeros/química , EletrodosRESUMO
BACKGROUND: Imaging and reconstruction of the morphology of neurons within the entire central nervous system (CNS) is important for deciphering the neural circuitry and related brain functions. With combination of tissue clearing and light sheet microscopy, previous studies have imaged the mouse CNS at cellular resolution, while remaining single axons unresolvable due to the tradeoff between sample size and imaging resolution. This could be improved by sectioning the sample into thick slices and imaged with high resolution light sheet microscopy as described in our previous study. However, the achievable quality for 3D imaging of serial thick slices is often hindered by surface undulation and other artifacts introduced by sectioning and handling limitations. NEW METHODS: In order to improve the imaging quality for mouse CNS, we develop a high-performance vibratome system for sample sectioning and handling automation. The sectioning mechanism of the system was modeled theoretically and verified experimentally. The effects of process parameters and sample properties on sectioning accuracy were studied to optimize the sectioning outcome. The resultant imaging outcome was demonstrated on mouse samples. RESULTS: Our theoretical model of vibratome effectively depicts the relationship between the sample surface undulation errors and the sectioning parameters. With the guidance of the theoretical model, the vibratome is able to achieve a local surface undulation error of ±0.5 µm and a surface arithmetic mean deviation (Sa) of 220 nm for 300-µm-thick tissue slices. Imaging results of mouse CNS show the continuous sectioning capability of the vibratome. COMPARISON WITH EXISTING METHOD: Our automatic sectioning and handling system is able to process serial thick slices for 3D imaging of the whole CNS at a single-axon resolution, superior to the commercially available vibratome devices. CONCLUSION: Our automatic sectioning and handling system can be optimized to prepare thick sample slices with minimal surface undulation and manual manipulation in support of 3D brain mapping with high-throughput and high-accuracy.
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Encéfalo , Imageamento Tridimensional , Camundongos , Animais , Imageamento Tridimensional/métodos , Encéfalo/anatomia & histologia , Vibração , Neurônios/fisiologia , Sistema Nervoso Central/diagnóstico por imagemRESUMO
Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.
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Encéfalo , Cálcio , Animais , Camundongos , Iluminação , Microscopia , FótonsRESUMO
In mitosis, accurate chromosome segregation depends on the kinetochore, a supermolecular machinery that couples dynamic spindle microtubules to centromeric chromatin. However, the structure-activity relationship of the constitutive centromere-associated network (CCAN) during mitosis remains uncharacterized. Building on our recent cryo-electron microscopic analyses of human CCAN structure, we investigated how dynamic phosphorylation of human CENP-N regulates accurate chromosome segregation. Our mass spectrometric analyses revealed mitotic phosphorylation of CENP-N by CDK1, which modulates the CENP-L-CENP-N interaction for accurate chromosome segregation and CCAN organization. Perturbation of CENP-N phosphorylation is shown to prevent proper chromosome alignment and activate the spindle assembly checkpoint. These analyses provide mechanistic insight into a previously undefined link between the centromere-kinetochore network and accurate chromosome segregation.
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Proteína Quinase CDC2 , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , Humanos , Proteína Quinase CDC2/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Mitose , FosforilaçãoRESUMO
Cooperation is a social behavior crucial for the survival of many species, including humans. Several experimental paradigms have been established to study cooperative behavior and related neural activity in different animal species. Although mice exhibit limited cooperative capacity in some behavioral paradigms, it is still interesting to explore their cooperative behavior and the underlying neural mechanisms. Here, we developed a new paradigm for training and testing cooperative behavior in mice based on coordinated lever-pressing and analyzed social interactions between the animals during cooperation. We observed extensive social contact and waiting behavior in cooperating animals, with the number of such events positively correlated with the success of cooperation. Using c-Fos immunostaining and a high-speed volumetric imaging with synchronized on-the-fly scan and readout (VISoR) system, we further mapped whole-brain neuronal activity trace following cooperation. Significantly higher levels of c-Fos expression were observed in cortical areas including the frontal pole, motor cortex, anterior cingulate area, and prelimbic area. These observations highlight social interaction and coordination in cooperative behavior and provide clues for further study of the underlying neural circuitry mechanisms.
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Comportamento Cooperativo , Comportamento Social , Humanos , Camundongos , Animais , Encéfalo/fisiologia , Aprendizagem , Giro do CínguloRESUMO
The nucleus accumbens (NAc) plays an important role in motivation and reward processing. Recent studies suggest that different NAc subnuclei differentially contribute to reward-related behaviors. However, how reward is encoded in individual NAc neurons remains unclear. Using in vivo single-cell resolution calcium imaging, we find diverse patterns of reward encoding in the medial and lateral shell subdivision of the NAc (NAcMed and NAcLat, respectively). Reward consumption increases NAcLat activity but decreases NAcMed activity, albeit with high variability among neurons. The heterogeneity in reward encoding could be attributed to differences in their synaptic inputs and transcriptional profiles. Specific optogenetic activation of Nts-positive neurons in the NAcLat promotes positive reinforcement, while activation of Cartpt-positive neurons in the NAcMed induces behavior aversion. Collectively, our study shows the organizational and transcriptional differences in NAc subregions and provides a framework for future dissection of NAc subregions in physiological and pathological conditions.
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Neurônios , Núcleo Accumbens , Núcleo Accumbens/fisiologia , Neurônios/fisiologia , Motivação , RecompensaRESUMO
Acetylcholine (ACh) is an important neuromodulator in various cognitive functions. However, it is unclear how ACh influences neural circuit dynamics by altering cellular properties. Here, we investigated how ACh influences reverberatory activity in cultured neuronal networks. We found that ACh suppressed the occurrence of evoked reverberation at low to moderate doses, but to a much lesser extent at high doses. Moreover, high doses of ACh caused a longer duration of evoked reverberation, and a higher occurrence of spontaneous activity. With whole-cell recording from single neurons, we found that ACh inhibited excitatory postsynaptic currents (EPSCs) while elevating neuronal firing in a dose-dependent manner. Furthermore, all ACh-induced cellular and network changes were blocked by muscarinic, but not nicotinic receptor antagonists. With computational modeling, we found that simulated changes in EPSCs and the excitability of single cells mimicking the effects of ACh indeed modulated the evoked network reverberation similar to experimental observations. Thus, ACh modulates network dynamics in a biphasic fashion, probably by inhibiting excitatory synaptic transmission and facilitating neuronal excitability through muscarinic signaling pathways.
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Acetilcolina , Colinérgicos , Colinérgicos/farmacologia , Acetilcolina/farmacologia , Acetilcolina/metabolismo , Neurônios/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Investigation of neural growth and connection is crucial in the field of neural tissue engineering. Here, using a femtosecond laser direct writing (fs-DLW) technique, we propose a directionally aligned porous microtube array as a culture system for accelerating the growth of neurons and directing the connection of neurites. These microtubes exhibited an unprecedented guidance effect toward the outgrowth of primary embryonic rat hippocampal neurons, with a wrap resembling the myelin sheaths of neurons. The speed of neurite growth inside these microtubes was significantly faster than that outside these microtubes. We also achieved selective/directing connection of neural networks inside the magnetic microtubes via precise microtube delivery to a gap between two neural clusters. This work not only proposes a powerful microtube platform for accelerated growth of neurons but also offers a new idea for constructing biological neural circuits by arranging the size, location, and pattern of microtubes.
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Neuritos , Neurônios , Animais , Ratos , Porosidade , Neurônios/fisiologia , Engenharia Tecidual , NeurogêneseRESUMO
Cryogenic electron tomography (cryoET) allows visualization of cellular structures in situ. However, anisotropic resolution arising from the intrinsic "missing-wedge" problem has presented major challenges in visualization and interpretation of tomograms. Here, we have developed IsoNet, a deep learning-based software package that iteratively reconstructs the missing-wedge information and increases signal-to-noise ratio, using the knowledge learned from raw tomograms. Without the need for sub-tomogram averaging, IsoNet generates tomograms with significantly reduced resolution anisotropy. Applications of IsoNet to three representative types of cryoET data demonstrate greatly improved structural interpretability: resolving lattice defects in immature HIV particles, establishing architecture of the paraflagellar rod in Eukaryotic flagella, and identifying heptagon-containing clathrin cages inside a neuronal synapse of cultured cells. Therefore, by overcoming two fundamental limitations of cryoET, IsoNet enables functional interpretation of cellular tomograms without sub-tomogram averaging. Its application to high-resolution cellular tomograms should also help identify differently oriented complexes of the same kind for sub-tomogram averaging.
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Aprendizado Profundo , Tomografia com Microscopia Eletrônica , Tomografia com Microscopia Eletrônica/métodos , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , SoftwareRESUMO
The pedunculopontine nucleus (PPN) is a heterogeneous midbrain structure involved in various brain functions, such as motor control, learning, reward, and sleep. Previous studies using conventional tracers have shown that the PPN receives extensive afferent inputs from various cortical areas. To examine how these cortical axons make collateral projections to other subcortical areas, we used a dual-viral injection strategy to sparsely label PPN-targeting cortical pyramidal neurons in CaMKIIα-Cre transgenic mice. Using a high-speed volumetric imaging with on-the-fly-scan and Readout (VISoR) technique, we visualized brain-wide axonal projections of individual PPN-targeting neurons from several cortical areas, including the prelimbic region (PL), anterior cingulate area (ACA) and secondary motor cortex (MOs). We found that each PPN-projecting neuron had a unique profile of collateralization, with some subcortical areas being preferential targets. In particular, PPN-projecting neurons from all three traced cortical areas exhibited common preferential collateralization to several nuclei, with most neurons targeting the striatum (STR), lateral hypothalamic area (LHA) and periaqueductal gray (PAG), and a substantial portion of neurons also targeting the zona incerta (ZI), median raphe nucleus (MRN) and substantia nigra pars reticulata (SNr). Meanwhile, very specific collateralization patterns were found for other nuclei, including the intermediate reticular nucleus (IRN), parvicellular reticular nucleus (PARN) and gigantocellular reticular nucleus (GRN), which receive collateral inputs almost exclusively from the MOs. These observations provide potential anatomical mechanisms for cortical neurons to coordinate the PPN with other subcortical areas in performing different physiological functions.