Rabies Virus Tracing Research Articles

Adaptation to photoperiod via dynamic neurotransmitter segregation.

Changes in the amount of daylight (photoperiod) alter physiology and behaviour1,2. Adaptive responses to seasonal photoperiods are vital to all organisms-dysregulation associates with disease, including affective disorders3 and metabolic syndromes4. The circadian rhythm circuitry is implicated in such responses5,6, yet little is known about the precise cellular substrates that underlie phase synchronization to photoperiod change. Here we identifya brain circuit and system of axon branch-specific and reversible neurotransmitter deployment that are critical for behavioural and sleep adaptation to photoperiod. A type of neuron called mrEn1-Pet17 in the mouse brainstem median raphe nucleus segregates serotonin from VGLUT3 (also known as SLC17A8,a proxy for glutamate) to different axonal branches that innervate specific brain regions involved in circadian rhythm and sleep-wake timing8,9. This branch-specific neurotransmitter deployment did not distinguish between daylight and dark phase; however, it reorganized with change in photoperiod. Axonal boutons, but not cell soma, changed neurochemical phenotype upon a shift away from equinox light/dark conditions, and these changes were reversed upon return to equinox conditions. When we genetically disabled Vglut3 in mrEn1-Pet1 neurons, sleep-wake periods, voluntary activity and clock gene expression did not synchronize to the new photoperiod or were delayed. Combining intersectional rabies virus tracing and projection-specific neuronal silencing, we delineated a preoptic area-to-mrEn1Pet1 connection that was responsible for decoding the photoperiodic inputs, driving the neurotransmitter reorganization and promoting behavioural synchronization. Our results reveal a brain circuit and periodic, branch-specific neurotransmitter deployment that regulates organismal adaptation to photoperiod change.

Electrophysiological Properties of Proprioception-Related Neurons in the Intermediate Thoracolumbar Spinal Cord.

Proprioception, the sense of limb and body position, is required to produce accurate and precise movements. Proprioceptive sensory neurons transmit muscle length and tension information to the spinal cord. The function of excitatory neurons in the intermediate spinal cord, which receive this proprioceptive information, remains poorly understood. Using genetic labeling strategies and patch-clamp techniques in acute spinal cord preparations in mice, we set out to uncover how two sets of spinal neurons, Clarke's column (CC) and Atoh1-lineage neurons, respond to electrical activity and how their inputs are organized. Both sets of neurons are located in close proximity in laminae V-VII of the thoracolumbar spinal cord and have been described to receive proprioceptive signals. We find that a majority of CC neurons have a tonic-firing type and express a distinctive hyperpolarization-activated current (Ih). Atoh1-lineage neurons, which cluster into two spatially distinct populations, are mostly a fading-firing type and display similar electrophysiological properties to each other, possibly due to their common developmental lineage. Finally, we find that CC neurons respond to stimulation of lumbar dorsal roots, consistent with prior knowledge that CC neurons receive hindlimb proprioceptive information. In contrast, using a combination of electrical stimulation, optogenetic stimulation, and transsynaptic rabies virus tracing, we find that Atoh1-lineage neurons receive heterogeneous, predominantly local thoracic inputs that include parvalbumin-lineage sensory afferents and local interneuron presynaptic inputs. Altogether, we find that CC and Atoh1-lineage neurons have distinct membrane properties and sensory input organization, representing different subcircuit modes of proprioceptive information processing.

Morphine- and foot shock-responsive neuronal ensembles in the VTA possess different connectivity and biased GPCR signaling pathway.

Background: Neurons in the ventral tegmental area (VTA) are sensitive to stress and their maladaptation have been implicated in the psychiatric disorders such as anxiety and addiction, etc. The cellular properties of the VTA neurons in response to different stressors related to different emotional processing remain to be investigated. Methods: By combining immediate early gene (IEG)-dependent labeling, rabies virus tracing, ensemble-specific transcriptomic analysis and fiber photometry recording in the VTA of male mice, the spatial distribution, brain-wide connectivity and cellular signaling pathways in the VTA neuronal ensembles in response to morphine (Mor-Ens) or foot shock (Shock-Ens) stimuli were investigated. Results: Optogenetic activation of the Mor-Ens drove approach behavior, whereas chemogenetic activation of the Shock-Ens increased the anxiety level in mice. Mor-Ens were clustered and enriched in the ventral VTA, contained a higher proportion of dopaminergic neurons, received more inputs from the dorsal medial striatum and the medial hypothalamic zone, and exhibited greater axonal arborization in the zona incerta and ventral pallidum. Whereas Shock-Ens were more dispersed, contained a higher proportion of GABAergic neurons, and received more inputs from the ventral pallidum and the lateral hypothalamic area. The downstream targets of the G protein and β-arrestin pathways, PLCβ3 and phosphorylated AKT1Thr308, were relatively enriched in the Mor-Ens and Shock-Ens, respectively. Cariprazine, the G-protein-biased agonist for the dopamine D2 receptor, increased the response of Mor-Ens to sucrose water and decreased the anxiety-like behavior during morphine withdrawal, whereas the β-arrestin-biased agonist UNC9994 decreased the response of Shock-Ens to tail suspension. Conclusions: Taken together, these findings reveal the heterogeneous connectivity and signaling pathways of the VTA neurons in response to morphine and foot shock, providing new insights for development of specific interventions for psychiatric disorders caused by various stressors associated with different VTA neuronal functions.

Mapping entorhinal cortex circuitry in mouse models of Alzheimer’s Disease

AbstractBackgroundAlzheimer’s disease (AD) is one of the most common neurodegenerative diseases worldwide that comes with significant personal and economic burdens. Unfortunately, the brain circuitry underlying AD pathophysiology is yet to be fully understood. Elucidating the circuits that change before the onset of cognitive deficits would help to identify neuronal populations that change early in disease progression. After the identification of these brain regions and cell types, they can be selectively targeted to reverse both the anatomical pathology and eventual behavioral decline.MethodHere we used a novel viral‐genetic method, rabies virus (RABV) tracing, which maps the inputs of targeted starter cell populations. We utilized this method in two mouse models of AD to measure circuit changes in the entorhinal cortex, which is one of the earliest brain regions to degenerate in Alzheimer’s. We implemented RABV mapping at three different time points, using aging as a key measure of comparison between circuit states. In using RABV tracing throughout disease progression, we were able to capture how these circuits look before any overt behavioral or histopathological deficits, as well as at a more advanced stage of the disease when both cognitive and pathological deficits are present.ResultOur preliminary results have identified key cells and circuits that are disrupted related to AD pathogenesis as targets to slow or prevent the development of the disease.ConclusionKey cells and circuits change early on in AD pathogenesis elucidated by rabies virus tracing.

Hippocampal neural circuit connectivity alterations in an Alzheimer's disease mouse model revealed by monosynaptic rabies virus tracing

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with growing major health impacts, particularly in countries with aging populations. The examination of neural circuit mechanisms in AD mouse models is a recent focus for identifying new AD treatment strategies. We hypothesize that age-progressive changes of both long-range and local hippocampal neural circuit connectivity occur in AD. Recent advancements in viral-genetic technologies provide new opportunities for semi-quantitative mapping of cell-type-specific neural circuit connections in AD mouse models. We applied a recently developed monosynaptic rabies tracing method to hippocampal neural circuit mapping studies in AD model mice to determine how local and global circuit connectivity to hippocampal CA1 excitatory neurons may be altered in the single APP knock-in (APP-KI) AD mouse model. To determine age-related AD progression, we measured circuit connectivity in age-matched littermate control and AD model mice at two different ages (3–4 vs. 10–11 months old). We quantitatively mapped the connectivity strengths of neural circuit inputs to hippocampal CA1 excitatory neurons from brain regions including hippocampal subregions, medial septum, subiculum and entorhinal cortex, comparing different age groups and genotypes. We focused on hippocampal CA1 because of its clear relationship with learning and memory and that the hippocampal formation shows clear neuropathological changes in human AD. Our results reveal alterations in circuit connectivity of hippocampal CA1 in AD model mice. Overall, we find weaker extrinsic CA1 input connectivity strengths in AD model mice compared with control mice, including sex differences of reduced subiculum to CA1 inputs in aged female AD mice compared with aged male AD mice. Unexpectedly, we find a connectivity pattern shift with an increased proportion of inputs from the CA3 region to CA1 excitatory neurons when comparing young and old AD model mice, as well as old wild-type mice and old AD model mice. These unexpected shifts in CA3-CA1 input proportions in this AD mouse model suggest the possibility that compensatory circuit increases may occur in response to connectivity losses in other parts of the hippocampal circuits. We expect that this work provides new insights into the neural circuit mechanisms of AD pathogenesis.

Brain injury environment critically influences the connectivity of transplanted neurons.

Cell transplantation is a promising approach for the reconstruction of neuronal circuits after brain damage. Transplanted neurons integrate with remarkable specificity into circuitries of the mouse cerebral cortex affected by neuronal ablation. However, it remains unclear how neurons perform in a local environment undergoing reactive gliosis, inflammation, macrophage infiltration, and scar formation, as in traumatic brain injury (TBI). To elucidate this, we transplanted cells from the embryonic mouse cerebral cortex into TBI-injured, inflamed-only, or intact cortex of adult mice. Brain-wide quantitative monosynaptic rabies virus (RABV) tracing unraveled graft inputs from correct regions across the brain in all conditions, with pronounced quantitative differences: scarce in intact and inflamed brain versus exuberant after TBI. In the latter, the initial overshoot is followed by pruning, with only a few input neurons persisting at 3 months. Proteomic profiling identifies candidate molecules for regulation of the synaptic yield, a pivotal parameter to tailor for functional restoration of neuronal circuits.

Excessive local host-graft connectivity in aging and amyloid-loaded brain.

Transplantation is a clinically relevant approach for brain repair, but much remains to be understood about influences of the disease environment on transplant connectivity. To explore the effect of amyloid pathology in Alzheimer’s disease (AD) and aging, we examined graft connectivity using monosynaptic rabies virus tracing in APP/PS1 mice and in 16- to 18-month-old wild-type (WT) mice. Transplanted neurons differentiated within 4 weeks and integrated well into the host visual cortex, receiving input from the appropriate brain regions for this area. Unexpectedly, we found a prominent several-fold increase in local inputs, in both amyloid-loaded and aged environments. State-of-the-art deep proteome analysis using mass spectrometry highlights complement system activation as a common denominator of environments promoting excessive local input connectivity. These data therefore reveal the key role of the host pathology in shaping the input connectome, calling for caution in extrapolating results from one pathological condition to another.

Monosynaptic rabies virus tracing from projection-targeted single neurons

A single neuron integrates inputs from thousands of presynaptic neurons to generate outputs. Circuit tracing using G-deleted rabies virus (RVΔG) vectors permits the brain-wide labeling of presynaptic inputs to targeted single neurons. However, the experimental procedures are complex, and the success rate of circuit labeling is low because of the lack of validation to increase the accuracy and efficiency of monosynaptic RVΔG tracing from targeted single neurons. We established an efficient RVΔG tracing method from projection target-defined single neurons using TVA950, a transmembrane isoform of TVA receptors, for initial viral infection. Presynaptic neurons were transsynaptically labeled from 80 % of the TVA950-expressing single starter neurons that survived after infection with EnvA-pseudotyped RVΔG in the adult mouse brain. We labeled single neuronal networks in the primary visual cortex (V1) and higher visual areas, namely the posteromedial area (PM) and anteromedial area (AM), as well as the single neuronal networks of PM-projecting V1 single neurons. Monosynaptic RVΔG tracing from projection-targeted single neurons revealed the input–output organization of single neuronal networks. Single-neuron network analysis based on RVΔG tracing will help dissect the heterogeneity of neural circuits and link circuit motifs and large-scale networks across scales, thereby clarifying information processing and circuit computation in the brain.

Axo-axonic cells in the amygdala regulate associative fear learning.

Activity and plasticity of excitatory neurons are tightly regulated by local inhibitory neurons in a spatiotemporal specific manner. Axo-axonic cells (AACs) are a unique type of inhibitory neurons that express parvalbumin and innervate the axon initial segment (AIS) of excitatory neurons. While their anatomical features have been identified, the genetical marker and functional role remain unclear. Here, we show that vasoactive intestinal peptide receptor 2 (Vipr2)-expressing inhibitory neurons in the basolateral amygdala (BLA) exhibit the anatomical and electrophysiological properties typical of AACs. Furthermore, using an AACs-specific labeling approach, we conducted in vivo fiber photometry recording and functional inhibition experiments in a cued fear conditioning test. The activity of AACs was increased for both conditioned stimulus (CS: tone) and unconditioned stimulus (US: foot-shock), and inhibition of GABA transmission of AACs impaired cued fear conditioning. These results suggest that AACs are important for memory acquisition. Finally, we employed projection-specific monosynaptic rabies virus tracing to identify the direct monosynaptic inputs cells to AACs in the BLA. This study provides new insights into the detailed functional roles of AACs in the BLA.

Engineered synaptic tools reveal localized cAMP signaling in synapse assembly.

The physiological mechanisms driving synapse formation are elusive. Although numerous signals are known to regulate synapses, it remains unclear which signaling mechanisms organize initial synapse assembly. Here, we describe new tools, referred to as "SynTAMs" for synaptic targeting molecules, that enable localized perturbations of cAMP signaling in developing postsynaptic specializations. We show that locally restricted suppression of postsynaptic cAMP levels or of cAMP-dependent protein-kinase activity severely impairs excitatory synapse formation without affecting neuronal maturation, dendritic arborization, or inhibitory synapse formation. In vivo, suppression of postsynaptic cAMP signaling in CA1 neurons prevented formation of both Schaffer-collateral and entorhinal-CA1/temporoammonic-path synapses, suggesting a general principle. Retrograde trans-synaptic rabies virus tracing revealed that postsynaptic cAMP signaling is required for continuous replacement of synapses throughout life. Given that postsynaptic latrophilin adhesion-GPCRs drive synapse formation and produce cAMP, we suggest that spatially restricted postsynaptic cAMP signals organize assembly of postsynaptic specializations during synapse formation.

Long-range monosynaptic inputs targeting apical and basal dendrites of primary motor cortex deep output neurons.

The primary motor cortex (M1) integrates various long-range signals from other brain regions for the learning and execution of goal-directed movements. How the different inputs target the distinct apical and basal dendrites of M1 pyramidal neurons is crucial in understanding the functions of M1, but the detailed connectivity pattern is still largely unknown. Here, by combining cre-dependent rabies virus tracing, layer-specific chemical retrograde tracing, optogenetic stimulation, and electrophysiological recording, we mapped all long-range monosynaptic inputs to M1 deep output neurons in layer 5 (L5) in mice. We revealed that most upstream areas innervate both dendritic compartments concurrently. These include the sensory cortices, higher motor cortices, sensory and motor thalamus, association cortices, as well as many subcortical nuclei. Furthermore, the dichotomous inputs arise mostly from spatially segregated neuronal subpopulations within an upstream nucleus, and even in the case of an individual cortical layer. Therefore, these input areas could serve as both feedforward and feedback sources albeit via different subpopulations. Taken together, our findings revealed a previously unknown and highly intricate synaptic input pattern of M1L5 neurons, which implicates that the dendritic computations carried out by these neurons during motor execution or learning are far more complicated than we currently understand.

Proximal and distal spinal neurons innervating multiple synergist and antagonist motor pools.

Motoneurons (MNs) control muscle contractions, and their recruitment by premotor circuits is tuned to produce accurate motor behaviours. To understand how these circuits coordinate movement across and between joints, it is necessary to understand whether spinal neurons pre-synaptic to motor pools have divergent projections to more than one MN population. Here, we used modified rabies virus tracing in mice to investigate premotor interneurons projecting to synergist flexor or extensor MNs, as well as those projecting to antagonist pairs of muscles controlling the ankle joint. We show that similar proportions of premotor neurons diverge to synergist and antagonist motor pools. Divergent premotor neurons were seen throughout the spinal cord, with decreasing numbers but increasing proportion with distance from the hindlimb enlargement. In the cervical cord, divergent long descending propriospinal neurons were found in contralateral lamina VIII, had large somata, were neither glycinergic, nor cholinergic, and projected to both lumbar and cervical MNs. We conclude that distributed spinal premotor neurons coordinate activity across multiple motor pools and that there are spinal neurons mediating co-contraction of antagonist muscles.

The relationship between birth timing, circuit wiring, and physiological response properties of cerebellar granule cells

Cerebellar granule cells (GrCs) are usually regarded as a uniform cell type that collectively expands the coding space of the cerebellum by integrating diverse combinations of mossy fiber inputs. Accordingly, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region have not been reported. The only known cellular property that distinguishes otherwise homogeneous GrCs is the correspondence between GrC birth timing and the depth of the molecular layer to which their axons project. To determine the role birth timing plays in GrC wiring and function, we developed genetic strategies to access early- and late-born GrCs. We initiated retrograde monosynaptic rabies virus tracing from control (birth timing unrestricted), early-born, and late-born GrCs, revealing the different patterns of mossy fiber input to GrCs in vermis lobule 6 and simplex, as well as to early- and late-born GrCs of vermis lobule 6: sensory and motor nuclei provide more input to early-born GrCs, while basal pontine and cerebellar nuclei provide more input to late-born GrCs. In vivo multidepth two-photon Ca2+ imaging of axons of early- and late-born GrCs revealed representations of diverse task variables and stimuli by both populations, with modest differences in the proportions encoding movement, reward anticipation, and reward consumption. Our results suggest neither organized parallel processing nor completely random organization of mossy fiber→GrC circuitry but instead a moderate influence of birth timing on GrC wiring and encoding. Our imaging data also provide evidence that GrCs can represent generalized responses to aversive stimuli, in addition to recently described reward representations.

Optimization of whole-brain rabies virus tracing technology for small cell populations

The lateral hypothalamus (LH) is critically involved in the regulation of homeostatic energy balance. Some neurons in the LH express receptors for leptin (LepRb), a hormone known to increase energy expenditure and decrease energy intake. However, the neuroanatomical inputs to LepRb-expressing LH neurons remain unknown. We used rabies virus tracing technology to map these inputs, but encountered non-specific tracing. To optimize this technology for a minor cell population (LepRb is not ubiquitously expressed in LH), we used LepRb-Cre mice and assessed how different titers of the avian tumor virus receptor A (TVA) helper virus affected rabies tracing efficiency and specificity. We found that rabies expression is dependent on TVA receptor expression, and that leakiness of TVA receptors is dependent on the titer of TVA virus used. We concluded that a titer of 1.0–3.0 × 107 genomic copies per µl of the TVA virus is optimal for rabies tracing. Next, we successfully applied modified rabies virus tracing technology to map inputs to LepRb-expressing LH neurons. We discovered that other neurons in the LH itself, the periventricular hypothalamic nucleus (Pe), the posterior hypothalamic nucleus (PH), the bed nucleus of the stria terminalis (BNST), and the paraventricular hypothalamic nucleus (PVN) are the most prominent input areas to LepRb-expressing LH neurons.

Brain‐wide mapping of presynaptic inputs to basolateral amygdala neurons

The basolateral amygdala (BLA), a region critical for emotional processing, is the limbic hub that is connected with various brain regions. BLA neurons are classified into different subtypes that exhibit differential projection patterns and mediate distinct emotional behaviors; however, little is known about their presynaptic input patterns. In this study, we employed projection-specific monosynaptic rabies virus tracing to identify the direct monosynaptic inputs to BLA subtypes. We found that each neuronal subtype receives long-range projection input from specific brain regions. In contrast to their specific axonal projection patterns, all BLA neuronal subtypes exhibited relatively similar input patterns. This anatomical organization supports the idea that the BLA is a central integrator that associates sensory information in different modalities with valence and sends associative information to behaviorally relevant brain regions.

Delineating the organization of projection neuron subsets in primary visual cortex with multiple fluorescent rabies virus tracing

The impressive functions of the brain rely on an extensive connectivity matrix between specific neurons, the architecture of which is frequently characterized by one brain nucleus/region connecting to multiple targets, either via collaterals of the same projection neuron or several, differentially specified neurons. Delineating the fine architecture of projection neuron subsets in a specific brain region could greatly facilitate its circuit, computational, and functional resolution. Here, we developed multiple fluorescent rabies viruses (RV) to delineate the fine organization of corticothalamic projection neuron subsets in the primary visual cortex (V1). By simultaneously retrograde labeling multiple distinct subsets of corticothalamic projection neurons in V1 from their target nuclei in thalamus (dLGN, LP, LD), we observed that V1-dLGN corticothalamic projection neurons were densely concentrated in layer VI, except for several sparsely scattered neurons in layer V, while V1-LP and V1-LD corticothalamic projection neurons were localized to both layers V and VI. Meanwhile, we observed a fraction of V1 corticothalamic projection neurons targeting two thalamic nuclei, which was further confirmed by fMOST whole-brain imaging. The multiple fluorescent RV tracing tools can be extensively applied to resolve the architecture of projection neuron subsets in certain brain regions, with a strong potential to delineate the computational and functional organization of these brain regions.

Developmental divergence of sensory stimulus representation in cortical interneurons

Vasocative-intestinal-peptide (VIP+) and somatostatin (SST+) interneurons are involved in modulating barrel cortex activity and perception during active whisking. Here we identify a developmental transition point of structural and functional rearrangements onto these interneurons around the start of active sensation at P14. Using in vivo two-photon Ca2+ imaging, we find that before P14, both interneuron types respond stronger to a multi-whisker stimulus, whereas after P14 their responses diverge, with VIP+ cells losing their multi-whisker preference and SST+ neurons enhancing theirs. Additionally, we find that Ca2+ signaling dynamics increase in precision as the cells and network mature. Rabies virus tracings followed by tissue clearing, as well as photostimulation-coupled electrophysiology reveal that SST+ cells receive higher cross-barrel inputs compared to VIP+ neurons at both time points. In addition, whereas prior to P14 both cell types receive direct input from the sensory thalamus, after P14 VIP+ cells show reduced inputs and SST+ cells largely shift to motor-related thalamic nuclei.

Increased Callosal Connectivity in Reeler Mice Revealed by Brain-Wide Input Mapping of VIP Neurons in Barrel Cortex.

The neocortex is composed of layers. Whether layers constitute an essential framework for the formation of functional circuits is not well understood. We investigated the brain-wide input connectivity of vasoactive intestinal polypeptide (VIP) expressing neurons in the reeler mouse. This mutant is characterized by a migration deficit of cortical neurons so that no layers are formed. Still, neurons retain their properties and reeler mice show little cognitive impairment. We focused on VIP neurons because they are known to receive strong long-range inputs and have a typical laminar bias toward upper layers. In reeler, these neurons are more dispersed across the cortex. We mapped the brain-wide inputs of VIP neurons in barrel cortex of wild-type and reeler mice with rabies virus tracing. Innervation by subcortical inputs was not altered in reeler, in contrast to the cortical circuitry. Numbers of long-range ipsilateral cortical inputs were reduced in reeler, while contralateral inputs were strongly increased. Reeler mice had more callosal projection neurons. Hence, the corpus callosum was larger in reeler as shown by structural imaging. We argue that, in the absence of cortical layers, circuits with subcortical structures are maintained but cortical neurons establish a different network that largely preserves cognitive functions.

A whole-brain connectivity map of mouse insular cortex.

The insular cortex (IC) plays key roles in emotional and regulatory brain functions and is affected across psychiatric diseases. However, the brain-wide connections of the mouse IC have not been comprehensively mapped. Here, we traced the whole-brain inputs and outputs of the mouse IC across its rostro-caudal extent. We employed cell-type-specific monosynaptic rabies virus tracings to characterize afferent connections onto either excitatory or inhibitory IC neurons, and adeno-associated viral tracings to label excitatory efferent axons. While the connectivity between the IC and other cortical regions was highly bidirectional, the IC connectivity with subcortical structures was often unidirectional, revealing prominent cortical-to-subcortical or subcortical-to-cortical pathways. The posterior and medial IC exhibited resembling connectivity patterns, while the anterior IC connectivity was distinct, suggesting two major functional compartments. Our results provide insights into the anatomical architecture of the mouse IC and thus a structural basis to guide investigations into its complex functions.

Septal Signaling Suppresses Seizures Through Stimulating Somatostatin Cells.

Direct Septum-Hippocampus Cholinergic Circuit Attenuates Seizure Through Driving Somatostatin Inhibition Wang Y, Wang Y, Xu C, Wang S, Tan N, Chen C, Chen L, Wu X, Fei F, Cheng H, Lin W, Qi Y, Chen B, Liang J, Zhao J, Xu Z, Guo Y, Zhang S, Li X, Zhou Y, Duan S, Chen Z. Biol Psychiatry. 2020;87(9):843-856. doi:10.1016/j.biopsych.2019.11.014.Background:Previous studies indicated the involvement of cholinergic neurons in seizure; however, the specific role of the medial septum (MS)–hippocampus cholinergic circuit in temporal lobe epilepsy (TLE) has not yet been completely elucidated.Methods:In the current study, we used magnetic resonance imaging and diffusion tensor imaging to characterize the pathological change of the MS-hippocampus circuit in 42 patients with TLE compared with 22 healthy volunteers. Using optogenetics and chemogenetics, combined with in vivo or in vitro electrophysiology and retrograde rabies virus tracing, we revealed a direct MS-hippocampus cholinergic circuit that potently attenuates seizure through driving somatostatin inhibition in animal TLE models.Results:We found that patients with TLE with hippocampal sclerosis showed a decrease of neuronal fiber connectivity of the MS-hippocampus compared with healthy people. In the mouse TLE model, MS cholinergic neurons ceased firing during hippocampal seizures. Optogenetic and chemogenetic activation of MS cholinergic neurons (but not glutamatergic or GABAergic [γ-aminobutyric acidergic] neurons) significantly attenuated hippocampal seizures, while specific inhibition promoted hippocampal seizures. Electrophysiology combined with modified rabies virus tracing studies showed that direct (but not indirect) MS-hippocampal cholinergic projections mediated the anti-seizure effect by preferentially targeting hippocampal GABAergic neurons. Furthermore, chemogenetic inhibition of hippocampal somatostatin-positive (rather than parvalbumin-positive) subtype of GABAergic neurons reversed the anti-seizure effect of the MS-hippocampus cholinergic circuit, which was mimicked by activating somatostatin-positive neurons.Conclusions:These findings underscore the notable anti-seizure role of the direct cholinergic MS-hippocampus circuit in TLE through driving the downstream somatostatin effector. This may provide a better understanding of the changes of the seizure circuit and the precise spatiotemporal control of epilepsy.”