Neurotransmitter classification from electron microscopy images at synaptic sites in Drosophila melanogaster.

High-resolution electron microscopy of nervous systems has enabled the reconstruction of synaptic connectomes. However, we do not know the synaptic sign for each connection (i.e., whether a connection is excitatory or inhibitory), which is implied by the released transmitter. We demonstrate that artificial neural networks can predict transmitter types for presynapses from electron micrographs: a network trained to predict six transmitters (acetylcholine, glutamate, GABA, serotonin, dopamine, octopamine) achieves an accuracy of 87% for individual synapses, 94% for neurons, and 91% for known cell types across a D. melanogaster whole brain. We visualize the ultrastructural features used for prediction, discovering subtle but significant differences between transmitter phenotypes. We also analyze transmitter distributions across the brain and find that neurons that develop together largely express only one fast-acting transmitter (acetylcholine, glutamate, or GABA). We hope that our publicly available predictions act as an accelerant for neuroscientific hypothesis generation for the fly.

Vision-dependent specification of cell types and function in the developing cortex.

The role of postnatal experience in sculpting cortical circuitry, while long appreciated, is poorly understood at the level of cell types. We explore this in the mouse primary visual cortex (V1) using single-nucleus RNA sequencing, visual deprivation, genetics, and functional imaging. We find that vision selectively drives the specification of glutamatergic cell types in upper layers (L) (L2/3/4), while deeper-layer glutamatergic, GABAergic, and non-neuronal cell types are established prior to eye opening. L2/3 cell types form an experience-dependent spatial continuum defined by the graded expression of ∼200 genes, including regulators of cell adhesion and synapse formation. One of these genes, Igsf9b, a vision-dependent gene encoding an inhibitory synaptic cell adhesion molecule, is required for the normal development of binocular responses in L2/3. In summary, vision preferentially regulates the development of upper-layer glutamatergic cell types through the regulation of cell-type-specific gene expression programs.

GABA-receptive microglia selectively sculpt developing inhibitory circuits.

Microglia, the resident immune cells of the brain, have emerged as crucial regulators of synaptic refinement and brain wiring. However, whether the remodeling of distinct synapse types during development is mediated by specialized microglia is unknown. Here, we show that GABA-receptive microglia selectively interact with inhibitory cortical synapses during a critical window of mouse postnatal development. GABA initiates a transcriptional synapse remodeling program within these specialized microglia, which in turn sculpt inhibitory connectivity without impacting excitatory synapses. Ablation of GABAB receptors within microglia impairs this process and leads to behavioral abnormalities. These findings demonstrate that brain wiring relies on the selective communication between matched neuronal and glial cell types.

Mechanism of gating and partial agonist action in the glycine receptor.

Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter binding. Neurotransmitters that produce maximum open channel probabilities (Po) are full agonists, whereas those that yield lower than maximum Po are partial agonists. Cys-loop receptors are an important class of neurotransmitter receptors, yet a structure-based understanding of the mechanism of partial agonist action has proven elusive. Here, we study the glycine receptor with the full agonist glycine and the partial agonists taurine and γ-amino butyric acid (GABA). We use electrophysiology to show how partial agonists populate agonist-bound, closed channel states and cryo-EM reconstructions to illuminate the structures of intermediate, pre-open states, providing insights into previously unseen conformational states along the receptor reaction pathway. We further correlate agonist-induced conformational changes to Po across members of the receptor family, providing a hypothetical mechanism for partial and full agonist action at Cys-loop receptors.

Aralar Sequesters GABA into Hyperactive Mitochondria, Causing Social Behavior Deficits.

Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. Here, we show that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. We identify the regulation of GABA availability by mitochondrial activity as a biologically relevant mechanism and demonstrate its contribution to social behavior. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. We identify Aralar as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases our understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions.

Functional Ontogeny of Hypothalamic Agrp Neurons in Neonatal Mouse Behaviors.

Hypothalamic Agrp neurons regulate food ingestion in adult mice. Whether these neurons are functional before animals start to ingest food is unknown. Here, we studied the functional ontogeny of Agrp neurons during breastfeeding using postnatal day 10 mice. In contrast to adult mice, we show that isolation from the nursing nest, not milk deprivation or ingestion, activated Agrp neurons. Non-nutritive suckling and warm temperatures blunted this effect. Using in vivo fiber photometry, neonatal Agrp neurons showed a rapid increase in activity upon isolation from the nest, an effect rapidly diminished following reunion with littermates. Neonates unable to release GABA from Agrp neurons expressed blunted emission of isolation-induced ultrasonic vocalizations. Chemogenetic overactivation of these neurons further increased emission of these ultrasonic vocalizations, but not milk ingestion. We uncovered important functional properties of hypothalamic Agrp neurons during mouse development, suggesting these neurons facilitate offspring-to-caregiver bonding.

Hyperactivity with Disrupted Attention by Activation of an Astrocyte Synaptogenic Cue.

Hyperactivity and disturbances of attention are common behavioral disorders whose underlying cellular and neural circuit causes are not understood. We report the discovery that striatal astrocytes drive such phenotypes through a hitherto unknown synaptic mechanism. We found that striatal medium spiny neurons (MSNs) triggered astrocyte signaling via γ-aminobutyric acid B (GABAB) receptors. Selective chemogenetic activation of this pathway in striatal astrocytes in vivo resulted in acute behavioral hyperactivity and disrupted attention. Such responses also resulted in upregulation of the synaptogenic cue thrombospondin-1 (TSP1) in astrocytes, increased excitatory synapses, enhanced corticostriatal synaptic transmission, and increased MSN action potential firing in vivo. All of these changes were reversed by blocking TSP1 effects. Our data identify a form of bidirectional neuron-astrocyte communication and demonstrate that acute reactivation of a single latent astrocyte synaptogenic cue alters striatal circuits controlling behavior, revealing astrocytes and the TSP1 pathway as therapeutic targets in hyperactivity, attention deficit, and related psychiatric disorders.

The Gut Microbiota Mediates the Anti-Seizure Effects of the Ketogenic Diet.

The ketogenic diet (KD) is used to treat refractory epilepsy, but the mechanisms underlying its neuroprotective effects remain unclear. Here, we show that the gut microbiota is altered by the KD and required for protection against acute electrically induced seizures and spontaneous tonic-clonic seizures in two mouse models. Mice treated with antibiotics or reared germ free are resistant to KD-mediated seizure protection. Enrichment of, and gnotobiotic co-colonization with, KD-associated Akkermansia and Parabacteroides restores seizure protection. Moreover, transplantation of the KD gut microbiota and treatment with Akkermansia and Parabacteroides each confer seizure protection to mice fed a control diet. Alterations in colonic lumenal, serum, and hippocampal metabolomic profiles correlate with seizure protection, including reductions in systemic gamma-glutamylated amino acids and elevated hippocampal GABA/glutamate levels. Bacterial cross-feeding decreases gamma-glutamyltranspeptidase activity, and inhibiting gamma-glutamylation promotes seizure protection in vivo. Overall, this study reveals that the gut microbiota modulates host metabolism and seizure susceptibility in mice.

GABA Signaling Stimulates ß Cell Regeneration in Diabetic Mice.

GABA and the antimalarial drug artemether, which acts on GABAergic pathways, can drive pancreatic cells with an α-cell phenotype toward a ß-cell-like phenotype. As reported in two papers (Ben-Othman et al. and Li et al.), these drugs can stimulate the production of sufficient numbers of new ß-like cells to reverse severe diabetes in mice.

Transcriptional Architecture of Synaptic Communication Delineates GABAergic Neuron Identity.

Understanding the organizational logic of neural circuits requires deciphering the biological basis of neuronal diversity and identity, but there is no consensus on how neuron types should be defined. We analyzed single-cell transcriptomes of a set of anatomically and physiologically characterized cortical GABAergic neurons and conducted a computational genomic screen for transcriptional profiles that distinguish them from one another. We discovered that cardinal GABAergic neuron types are delineated by a transcriptional architecture that encodes their synaptic communication patterns. This architecture comprises 6 categories of ∼40 gene families, including cell-adhesion molecules, transmitter-modulator receptors, ion channels, signaling proteins, neuropeptides and vesicular release components, and transcription factors. Combinatorial expression of select members across families shapes a multi-layered molecular scaffold along the cell membrane that may customize synaptic connectivity patterns and input-output signaling properties. This molecular genetic framework of neuronal identity integrates cell phenotypes along multiple axes and provides a foundation for discovering and classifying neuron types.

Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity.

Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.

Decoding neural circuits that control compulsive sucrose seeking.

The lateral hypothalamic (LH) projection to the ventral tegmental area (VTA) has been linked to reward processing, but the computations within the LH-VTA loop that give rise to specific aspects of behavior have been difficult to isolate. We show that LH-VTA neurons encode the learned action of seeking a reward, independent of reward availability. In contrast, LH neurons downstream of VTA encode reward-predictive cues and unexpected reward omission. We show that inhibiting the LH-VTA pathway reduces "compulsive" sucrose seeking but not food consumption in hungry mice. We reveal that the LH sends excitatory and inhibitory input onto VTA dopamine (DA) and GABA neurons, and that the GABAergic projection drives feeding-related behavior. Our study overlays information about the type, function, and connectivity of LH neurons and identifies a neural circuit that selectively controls compulsive sugar consumption, without preventing feeding necessary for survival, providing a potential target for therapeutic interventions for compulsive-overeating disorder.

GABA blocks pathological but not acute TRPV1 pain signals.

Sensitization of the capsaicin receptor TRPV1 is central to the initiation of pathological forms of pain, and multiple signaling cascades are known to enhance TRPV1 activity under inflammatory conditions. How might detrimental escalation of TRPV1 activity be counteracted? Using a genetic-proteomic approach, we identify the GABAB1 receptor subunit as bona fide inhibitor of TRPV1 sensitization in the context of diverse inflammatory settings. We find that the endogenous GABAB agonist, GABA, is released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism limiting TRPV1 sensitization. The effect of GABAB on TRPV1 is independent of canonical G protein signaling and rather relies on close juxtaposition of the GABAB1 receptor subunit and TRPV1. Activating the GABAB1 receptor subunit does not attenuate normal functioning of the capsaicin receptor but exclusively reverts its sensitized state. Thus, harnessing this mechanism for anti-pain therapy may prevent adverse effects associated with currently available TRPV1 blockers.

Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic Input-Output Mapping.

Dopamine (DA) neurons in the midbrain ventral tegmental area (VTA) integrate complex inputs to encode multiple signals that influence motivated behaviors via diverse projections. Here, we combine axon-initiated viral transduction with rabies-mediated trans-synaptic tracing and Cre-based cell-type-specific targeting to systematically map input-output relationships of VTA-DA neurons. We found that VTA-DA (and VTA-GABA) neurons receive excitatory, inhibitory, and modulatory input from diverse sources. VTA-DA neurons projecting to different forebrain regions exhibit specific biases in their input selection. VTA-DA neurons projecting to lateral and medial nucleus accumbens innervate largely non-overlapping striatal targets, with the latter also sending extensive extra-striatal axon collaterals. Using electrophysiology and behavior, we validated new circuits identified in our tracing studies, including a previously unappreciated top-down reinforcing circuit from anterior cortex to lateral nucleus accumbens via VTA-DA neurons. This study highlights the utility of our viral-genetic tracing strategies to elucidate the complex neural substrates that underlie motivated behaviors.

Visualizing hypothalamic network dynamics for appetitive and consummatory behaviors.

Optimally orchestrating complex behavioral states, such as the pursuit and consumption of food, is critical for an organism's survival. The lateral hypothalamus (LH) is a neuroanatomical region essential for appetitive and consummatory behaviors, but whether individual neurons within the LH differentially contribute to these interconnected processes is unknown. Here, we show that selective optogenetic stimulation of a molecularly defined subset of LH GABAergic (Vgat-expressing) neurons enhances both appetitive and consummatory behaviors, whereas genetic ablation of these neurons reduced these phenotypes. Furthermore, this targeted LH subpopulation is distinct from cells containing the feeding-related neuropeptides, melanin-concentrating hormone (MCH), and orexin (Orx). Employing in vivo calcium imaging in freely behaving mice to record activity dynamics from hundreds of cells, we identified individual LH GABAergic neurons that preferentially encode aspects of either appetitive or consummatory behaviors, but rarely both. These tightly regulated, yet highly intertwined, behavioral processes are thus dissociable at the cellular level.

Network-level encoding of local neurotransmitters in cortical astrocytes.

Astrocytes, the most abundant non-neuronal cell type in the mammalian brain, are crucial circuit components that respond to and modulate neuronal activity through calcium (Ca2+) signalling1-7. Astrocyte Ca2+ activity is highly heterogeneous and occurs across multiple spatiotemporal scales-from fast, subcellular activity3,4 to slow, synchronized activity across connected astrocyte networks8-10-to influence many processes5,7,11. However, the inputs that drive astrocyte network dynamics remain unclear. Here we used ex vivo and in vivo two-photon astrocyte imaging while mimicking neuronal neurotransmitter inputs at multiple spatiotemporal scales. We find that brief, subcellular inputs of GABA and glutamate lead to widespread, long-lasting astrocyte Ca2+ responses beyond an individual stimulated cell. Further, we find that a key subset of Ca2+ activity-propagative activity-differentiates astrocyte network responses to these two main neurotransmitters, and may influence responses to future inputs. Together, our results demonstrate that local, transient neurotransmitter inputs are encoded by broad cortical astrocyte networks over a minutes-long time course, contributing to accumulating evidence that substantial astrocyte-neuron communication occurs across slow, network-level spatiotemporal scales12-14. These findings will enable future studies to investigate the link between specific astrocyte Ca2+ activity and specific functional outputs, which could build a consistent framework for astrocytic modulation of neuronal activity.

Antagonistic control of social versus repetitive self-grooming behaviors by separable amygdala neuronal subsets.

Animals display a range of innate social behaviors that play essential roles in survival and reproduction. While the medial amygdala (MeA) has been implicated in prototypic social behaviors such as aggression, the circuit-level mechanisms controlling such behaviors are not well understood. Using cell-type-specific functional manipulations, we find that distinct neuronal populations in the MeA control different social and asocial behaviors. A GABAergic subpopulation promotes aggression and two other social behaviors, while neighboring glutamatergic neurons promote repetitive self-grooming, an asocial behavior. Moreover, this glutamatergic subpopulation inhibits social interactions independently of its effect to promote self-grooming, while the GABAergic subpopulation inhibits self-grooming, even in a nonsocial context. These data suggest that social versus repetitive asocial behaviors are controlled in an antagonistic manner by inhibitory versus excitatory amygdala subpopulations, respectively. These findings provide a framework for understanding circuit-level mechanisms underlying opponency between innate behaviors, with implications for their perturbation in psychiatric disorders.

Norspermidine is not a self-produced trigger for biofilm disassembly.

Formation of Bacillus subtilis biofilms, consisting of cells encapsulated within an extracellular matrix of exopolysaccharide and protein, requires the polyamine spermidine. A recent study reported that (1) related polyamine norspermidine is synthesized by B. subtilis using the equivalent of the Vibrio cholerae biosynthetic pathway, (2) exogenous norspermidine at 25 µM prevents B. subtilis biofilm formation, (3) endogenous norspermidine is present in biofilms at 50-80 µM, and (4) norspermidine prevents biofilm formation by condensing biofilm exopolysaccharide. In contrast, we find that, at concentrations up to 200 µM, exogenous norspermidine promotes biofilm formation. We find that norspermidine is absent in wild-type B. subtilis biofilms at all stages, and higher concentrations of exogenous norspermidine eventually inhibit planktonic growth and biofilm formation in an exopolysaccharide-independent manner. Moreover, orthologs of the V. cholerae norspermidine biosynthetic pathway are absent from B. subtilis, confirming that norspermidine is not physiologically relevant to biofilm function in this species.

Sensing of amino acids in a dopaminergic circuitry promotes rejection of an incomplete diet in Drosophila.

The brain is the central organizer of food intake, matching the quality and quantity of the food sources with organismal needs. To ensure appropriate amino acid balance, many species reject a diet lacking one or several essential amino acids (EAAs) and seek out a better food source. Here, we show that, in Drosophila larvae, this behavior relies on innate sensing of amino acids in dopaminergic (DA) neurons of the brain. We demonstrate that the amino acid sensor GCN2 acts upstream of GABA signaling in DA neurons to promote avoidance of the EAA-deficient diet. Using real-time calcium imaging in larval brains, we show that amino acid imbalance induces a rapid and reversible activation of three DA neurons that are necessary and sufficient for food rejection. Taken together, these data identify a central amino-acid-sensing mechanism operating in specific DA neurons and controlling food intake.

A cortical circuit for gain control by behavioral state.

The brain's response to sensory input is strikingly modulated by behavioral state. Notably, the visual response of mouse primary visual cortex (V1) is enhanced by locomotion, a tractable and accessible example of a time-locked change in cortical state. The neural circuits that transmit behavioral state to sensory cortex to produce this modulation are unknown. In vivo calcium imaging of behaving animals revealed that locomotion activates vasoactive intestinal peptide (VIP)-positive neurons in mouse V1 independent of visual stimulation and largely through nicotinic inputs from basal forebrain. Optogenetic activation of VIP neurons increased V1 visual responses in stationary awake mice, artificially mimicking the effect of locomotion, and photolytic damage of VIP neurons abolished the enhancement of V1 responses by locomotion. These findings establish a cortical circuit for the enhancement of visual response by locomotion and provide a potential common circuit for the modulation of sensory processing by behavioral state.