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Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival1,2. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms3-5. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. Here we show that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. Our findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies.

Advanced imaging methods now allow cell-type-specific recording of neural activity across the mammalian brain, potentially enabling the exploration of how brain-wide dynamical patterns give rise to complex behavioural states1–12. Dissociation is an altered behavioural state in which the integrity of experience is disrupted, resulting in reproducible cognitive phenomena including the dissociation of stimulus detection from stimulus-related affective responses. Dissociation can occur as a result of trauma, epilepsy or dissociative drug use13,14, but despite its substantial basic and clinical importance, the underlying neurophysiology of this state is unknown. Here we establish such a dissociation-like state in mice, induced by precisely-dosed administration of ketamine or phencyclidine. Large-scale imaging of neural activity revealed that these dissociative agents elicited a 1–3-Hz rhythm in layer 5 neurons of the retrosplenial cortex. Electrophysiological recording with four simultaneously deployed high-density probes revealed rhythmic coupling of the retrosplenial cortex with anatomically connected components of thalamus circuitry, but uncoupling from most other brain regions was observed—including a notable inverse correlation with frontally projecting thalamic nuclei. In testing for causal significance, we found that rhythmic optogenetic activation of retrosplenial cortex layer 5 neurons recapitulated dissociation-like behavioural effects. Local retrosplenial hyperpolarization-activated cyclic-nucleotide-gated potassium channel 1 (HCN1) pacemakers were required for systemic ketamine to induce this rhythm and to elicit dissociation-like behavioural effects. In a patient with focal epilepsy, simultaneous intracranial stereoencephalography recordings from across the brain revealed a similarly localized rhythm in the homologous deep posteromedial cortex that was temporally correlated with pre-seizure self-reported dissociation, and local brief electrical stimulation of this region elicited dissociative experiences. These results identify the molecular, cellular and physiological properties of a conserved deep posteromedial cortical rhythm that underlies states of dissociation. Dissociative states in mouse and human brains are traced to low-frequency rhythmic neural activity—with distinct molecular, cellular and physiological properties—in the deep retrosplenial cortex and the posteromedial cortex.

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.

General principles of neuronal communication The explosion of modern technologies over the past years has produced vast amounts of knowledge about the molecular, anatomical, and physiological properties of individual neurons. However, individual neurons do not function in isolation; they interact with each other in circuits to process information. Luo attempted to synthesize different types of investigations to extract generalized principles underlying how neurons communicate with each other through specific patterns of synaptic connectivity. Although different elements of the projection motifs and architectural plans have been discussed separately in the past, these motifs and architectures can now be brought together in the same intellectual framework. —PRS

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 Ca(2+) 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.

Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. Here, we use the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 28 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations, and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics- corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibit “exploring branches” consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral interactions in contralateral target selection, and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.

Our understanding of the mechanisms of neural circuit assembly is far from complete. Identification of wiring molecules with novel mechanisms of action will provide insights into how complex and heterogeneous neural circuits assemble during development. In the Drosophila olfactory system, 50 classes of olfactory receptor neurons (ORNs) make precise synaptic connections with 50 classes of partner projection neurons (PNs). Here, we performed an RNA interference screen for cell surface molecules and identified the leucine-rich repeat–containing transmembrane protein known as Fish-lips (Fili) as a novel wiring molecule in the assembly of the Drosophila olfactory circuit. Fili contributes to the precise axon and dendrite targeting of a small subset of ORN and PN classes, respectively. Cell-type–specific expression and genetic analyses suggest that Fili sends a transsynaptic repulsive signal to neurites of nonpartner classes that prevents their targeting to inappropriate glomeruli in the antennal lobe.

Memories of fearful events can last a lifetime. The prelimbic (PL) cortex, a subregion of prefrontal cortex, plays a critical role in fear memory retrieval over time. Most studies have focused on acquisition, consolidation, and retrieval of recent memories, but much less is known about the neural mechanisms of remote memory. Using a new knock-in mouse for activity-dependent genetic labeling (TRAP2), we demonstrate that neuronal ensembles in the PL cortex are dynamic. PL neurons TRAPed during later memory retrievals are more likely to be reactivated and make larger behavioral contributions to remote memory retrieval compared to those TRAPed during learning or early memory retrieval. PL activity during learning is required to initiate this time-dependent reorganization in PL ensembles underlying memory retrieval. Finally, while neurons TRAPed during earlier and later retrievals have similar broad projections throughout the brain, PL neurons TRAPed later have a stronger functional recruitment of cortical targets.

A close view of the paraventricular thalamus The paraventricular thalamus is a relay station connecting brainstem and hypothalamic signals that represent internal states with the limbic forebrain that performs associative functions in emotional contexts. Zhu et al. found that paraventricular thalamic neurons represent multiple salient features of sensory stimuli, including reward, aversiveness, novelty, and surprise. The nucleus thus provides context-dependent salience encoding. The thalamus gates sensory information and contributes to the sleep-wake cycle through its interactions with the cerebral cortex. Ren et al. recorded from neurons in the paraventricular thalamus and observed that both population and single-neuron activity were tightly coupled with wakefulness. Science , this issue p. 423 , p. 429

Significance Losing one copy of the RAI1 gene causes Smith–Magenis syndrome (SMS), a neurodevelopmental disorder. Using a newly generated SMS mouse model, this study demonstrates that restoring the Rai1 gene dose in an early postnatal window could repair gene expression and social interaction deficits in this SMS model. The SMS mouse model also showed a reduced density of dendritic spines, anatomical correlates of excitatory synapses, in the prefrontal cortex. Artificial activation of prefrontal cortex neurons partially alleviated the behavioral deficits. These findings suggest that, similar to Rett syndrome, SMS is caused by disruption of a chromatin-modifying gene with reversible developmental phenotypes, highlighting the potential treatment windows in childhood or adolescence., Haploinsufficiency of Retinoic Acid Induced 1 (RAI1) causes Smith–Magenis syndrome (SMS), a syndromic autism spectrum disorder associated with craniofacial abnormalities, intellectual disability, and behavioral problems. There is currently no cure for SMS. Here, we generated a genetic mouse model to determine the reversibility of SMS-like neurobehavioral phenotypes in Rai1 heterozygous mice. We show that normalizing the Rai1 level 3–4 wk after birth corrected the expression of genes related to neural developmental pathways and fully reversed a social interaction deficit caused by Rai1 haploinsufficiency. In contrast, Rai1 reactivation 7–8 wk after birth was not beneficial. We also demonstrated that the correct Rai1 dose is required in both excitatory and inhibitory neurons for proper social interactions. Finally, we found that Rai1 heterozygous mice exhibited a reduction of dendritic spines in the medial prefrontal cortex (mPFC) and that optogenetic activation of mPFC neurons in adults improved the social interaction deficit of Rai1 heterozygous mice. Together, these results suggest the existence of a postnatal temporal window during which restoring Rai1 can improve the transcriptional and social behavioral deficits in a mouse model of SMS. It is possible that circuit-level interventions would be beneficial beyond this critical window.

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 properties that distinguishes otherwise homogeneous GrCs is the correspondence between GrC birthtime and the depth of the molecular layer to which their axons (parallel fibers) 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, 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 vivomulti-depth 2-photon Ca2+imaging of parallel fibers of early- and late-born GrCs revealed representations of diverse task variables and stimuli by both populations, with differences in the proportions of parallel fibers 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 suggest that GrCs can represent general aversiveness, in addition to recently described reward representations.Significance StatementCerebellar granule cells (GrCs) comprise the majority of all neurons in the mammalian brain and are usually regarded as a uniform cell type. However, the birth timing of an individual GrC dictates where its axon projects. Using viral-genetic techniques, we find that early- and late-born GrCs receive different proportions of inputs from the same set of input regions. Usingin vivomulti-depth 2-photon Ca2+imaging of axons of early- and late-born GrCs, we found that both populations represent diverse task variables and stimuli, with differences in the proportions of axons in encoding of a subset of movement and reward parameters. These results indicate that birth timing contributes to the input selection and physiological response properties of GrCs.

Recognition of environmental cues is essential for the survival of all organisms. Transcriptional changes occur to enable the generation and function of the neural circuits underlying sensory perception. To gain insight into these changes, we generated single-cell transcriptomes ofDrosophilaolfactory- (ORNs), thermo-, and hygro-sensory neurons at an early developmental and adult stage using single-cell and single-nucleus RNA sequencing. We discovered that ORNs maintain expression of the same olfactory receptors across development. Using receptor expression and computational approaches, we matched transcriptomic clusters corresponding to anatomically and physiologically defined neuron types across multiple developmental stages. We found that cell-type-specific transcriptomes partly reflected axon trajectory choices in development and sensory modality in adults. We uncovered stage-specific genes that could regulate the wiring and sensory responses of distinct ORN types. Collectively, our data reveal transcriptomic features of sensory neuron biology and provide a resource for future studies of their development and physiology.

Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological properties. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes ofDrosophilaolfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage—neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.

Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. Here, we use the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 28 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations, and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics-corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibit “exploring branches” consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral interactions in contralateral target selection, and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.

INTRODUCTION: The brains of extant animals have evolved over hundreds of millions of years from simple circuits. Cell types diversified, connections elaborated, and new brain regions emerged. Models for brain region evolution range from duplication of existing regions to splitting of previously multifunctional regions and de novo assembly from existing cell types. These models, however, have not been demonstrated in vertebrate brains at cell-type resolution. RATIONALE: We investigated brain region evolution using the cerebellar nuclei as a model system. The cerebellum is a major hindbrain structure in jawed vertebrates, comprising the cerebellar cortex and cerebellar nuclei. It is thought to act as a feedforward model for motor control and cognitive processes. The cerebellar cortex receives and processes inputs and sends outputs to the cerebellar nuclei, which route the results of cerebellar computations to the rest of the brain. Whereas the cerebellar cortex is well conserved across vertebrates, the cerebellar nuclei vary in number, with none in jawless vertebrates, one pair in cartilaginous fishes and amphibians, two pairs in reptiles and birds, and three pairs in mammals. This pattern suggests that extant cerebellar nuclei evolved from a single ancestral nucleus. Cerebellar nuclei thus provide a good model to interrogate brain region evolution. RESULTS: We characterized the cerebellar nuclei in mice, chickens, and humans using whole-brain and spinal cord projection mapping in cleared samples, single-nucleus RNA sequencing (snRNAseq), and spatially resolved transcript amplicon readout mapping (STARmap) analysis. We first compared the projection patterns of the three cerebellar nuclei of mice. Our data reveal broad projections of all nuclei, which in common target regions are shifted relative to each other. To understand the transcriptomic differences that underlie these shifting projections, we produced a cell-type atlas of the mouse cerebellar nuclei using snRNAseq. We discovered three region-invariant inhibitory cell classes and 15 region-specific excitatory cell types. Excitatory cell types fall into two classes with distinct gene expression and electrophysiological properties. Members of each class are present in every nucleus and are putative sister cell types. STARmap analysis in mice revealed that the organizational unit of the cerebellar nuclei is cytoarchitectonically distinguishable subnuclei, each of which contains the three inhibitory and two excitatory classes. To test whether this archetypal subnucleus is also the evolutionary unit of the cerebellar nuclei, we performed snRNAseq and STARmap on the chicken cerebellar nuclei. We identified four subnuclei, three of which had direct orthologs in mice. Each chicken subnucleus contained the same cell-type set of three inhibitory and two excitatory classes already identified in mice, confirming our hypothesis. Cerebellar nuclei vary in size across vertebrates. In particular, the human lateral nucleus is markedly expanded. To understand this expansion, we performed snRNAseq in humans. We found that the medial and interposed nuclei maintained the archetypal cerebellar nuclei composition. However, the lateral nucleus expanded one excitatory cell class at the expense of the other. Conditional tracing in the mouse lateral nucleus revealed that the cell class expanded in humans preferentially accesses lateral frontal cortices via specific intermediate thalamic nuclei. CONCLUSION: We identified a conserved cell-type set that forms an archetypal cerebellar nucleus as the unit of cerebellar nuclei organization and evolution. We propose that this archetypal nucleus was repeatedly duplicated during evolution, accompanied primarily by transcriptomic divergence of excitatory neurons and shifts in their projection patterns. Our data support a model of duplication-and-divergence of entire cell-type sets for brain region evolution.

Recognition of environmental cues is essential for the survival of all organisms. Precise transcriptional changes occur to enable the generation and function of the neural circuits underlying sensory perception. To gain insight into these changes, we generated single-cell transcriptomes ofDrosophilaolfactory receptor neurons (ORNs), thermosensory and hygrosensory neurons from the third antennal segment at an early developmental and adult stage. We discovered that ORNs maintain expression of the same olfactory receptors across development. Using these receptors and computational approaches, we matched transcriptomic clusters corresponding to anatomically and physiologically defined neuronal types across multiple developmental stages. Cell-type-specific transcriptomes, in part, reflected axon trajectory choices in early development and sensory modality in adults. Our analysis also uncovered type-specific and broadly expressed genes that could modulate adult sensory responses. Collectively, our data reveal important transcriptomic features of sensory neuron biology and provides a resource for future studies of their development and physiology.

Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological properties. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of Drosophila olfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage—neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.

Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological features. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes ofDrosophilaolfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage—neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.

Large scientific projects in genomics and astronomy are influential not because they answer any single question but because they enable investigation of continuously arising new questions from the same data-rich sources. Advances in automated mapping of the brain's synaptic connections (connectomics) suggest that the complicated circuits underlying brain function are ripe for analysis. We discuss benefits of mapping a mouse brain at the level of synapses.

Dopamine neurons project to diverse regions throughout the brain to modulate various brain processes and behaviors. It is increasingly appreciated that dopamine neurons are heterogeneous in their gene expression, circuitry, physiology, and function. Current approaches to target dopamine neurons are largely based on single gene drivers, which either label all dopamine neurons, or mark a sub-set but concurrently label non-dopaminergic neurons. Here we establish a novel mouse line in which Flp recombinase is knocked-in to the endogenousSlc6a3(dopamine active transporter, DAT) locus. DAT-Flp mice can be used with various Cre-expressing mouse lines to efficiently and selectively label dopaminergic subpopulations using Cre/Flp-dependent intersectional strategies. We demonstrate the utility of this approach by crossing DAT-Flp mice with NEX-Cre mice, to specifically labelNeurod6-expressing dopamine neurons that project to the nucleus accumbens medial shell. DAT-Flp mice represent a novel tool, which will help parse the diverse functions mediated by dopaminergic circuits.

The synaptotrophic hypothesis posits that synapse formation stabilizes dendritic branches, but this hypothesis has not been causally tested in vivo in the mammalian brain. The presynaptic ligand cerebellin-1 (Cbln1) and postsynaptic receptor GluD2 mediate synaptogenesis between granule cells and Purkinje cells in the molecular layer of the cerebellar cortex. Here we show that sparse but not global knockout of GluD2 causes under-elaboration of Purkinje cell dendrites in the deep molecular layer and overelaboration in the superficial molecular layer. Developmental, overexpression, structure-function, and genetic epistasis analyses indicate that these dendrite morphogenesis defects result from a deficit in Cbln1/GluD2-dependent competitive interactions. A generative model of dendrite growth based on competitive synaptogenesis largely recapitulates GluD2 sparse and global knockout phenotypes. Our results support the synaptotrophic hypothesis at initial stages of dendrite development, suggest a second mode in which cumulative synapse formation inhibits further dendrite growth, and highlight the importance of competition in dendrite morphogenesis.

Mother-infant bonding develops rapidly following parturition and is accompanied by changes in sensory perception and behavior. Here, we studied how ultrasonic vocalizations (USVs) are represented in the brain of mothers. Using a mouse line that allows temporally controlled genetic access to active neurons, we found that the temporal association cortex (TeA) in mothers exhibits robust USV responses. Rabies tracing from USV-responsive neurons revealed extensive subcortical and cortical inputs into TeA. A particularly dominant cortical source of inputs is primary auditory cortex (A1), suggesting strong A1-to-TeA connectivity. Chemogenetic silencing of USV-responsive neurons in TeA impaired auditory-driven maternal preference in a pup-retrieval assay. Furthermore, dense extracellular recordings from awake mice revealed changes of both single neurons’ and population responses to USVs in TeA; improving discriminability of pup calls in mothers as compared to naïve females. These data indicate that TeA plays a key role in encoding and perceiving pup cries during motherhood.

Parallel information processing is a salient feature of complex nervous systems. For example, the medial and lateral hippocampal networks (MHN and LHN) preferentially process spatial- and object-related information, respectively. However, the mechanisms underlying parallel network assembly during development remain largely unknown. Here, we show that complementary expression of cell-surface molecules Teneurin-3 (Ten3) and Latrophilin-2 (Lphn2) in the MHN and LHN, respectively, guides the precise assembly of both the MHN and LHN. Viral-genetic perturbations in vivo demonstrate that Ten3+ axons are repelled by target-derived Lphn2, revealing that Lphn2/Ten3-mediated repulsion and Ten3/Ten3-mediated attraction cooperate to control precise target selection of MHN axons. In the LHN, Lphn2+ axons are confined to Lphn2+ targets via repulsion from Ten3+ targets. Our findings demonstrate that assembly of parallel hippocampal networks follows a ‘Ten3→Ten3, Lphn2→Lphn2’ rule instructed by reciprocal repulsions.

Significance Autism is a complex neurodevelopmental disorder whose causative mechanisms are unclear. Taking advantage of a unique cohort with recessively inherited autism, we identified six families with biallelic mutation of the neuronal-specific subunit of the BAF complex, ACTL6B (also known as BAF53b). Relative to all other genes, ACTL6B was the most statistically significant mutated gene in the recessive autism cohort. We describe autism-relevant phenotypes in human brain organoids and in mouse and fly models. We foresee the outcomes from this study will be the following: 1) a link between neuronal activity-dependent transcriptional repression and autism; 2) a characterization of mouse and fly models to study ACTL6B mutant autism; and 3) an understanding the role of ACTL6B and nBAF complexes in neuronal transcriptional regulation., Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such “early activation” genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors (Fos, Fosl2, Fosb, and Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism.

Emerging evidence supports the hypothesis that pathogenic protein aggregates associated with neurodegenerative diseases spread from cell to cell through the brain in a manner akin to infectious prions. Here, we show that mutant huntingtin (mHtt) aggregates associated with Huntington disease transfer anterogradely from presynaptic to postsynaptic neurons in the adult Drosophila olfactory system. Trans-synaptic transmission of mHtt aggregates is inversely correlated with neuronal activity and blocked by inhibiting caspases in presynaptic neurons, implicating synaptic dysfunction and cell death in aggregate spreading. Remarkably, mHtt aggregate transmission across synapses requires the glial scavenger receptor Draper and involves a transient visit to the glial cytoplasm, indicating that phagocytic glia act as obligatory intermediates in aggregate spreading between synaptically-connected neurons. These findings expand our understanding of phagocytic glia as double-edged players in neurodegeneration—by clearing neurotoxic protein aggregates, but also providing an opportunity for prion-like seeds to evade phagolysosomal degradation and propagate further in the brain.

SUMMARYSingle-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFC→PAG (periaqueductal gray). By comparing Ca2+-activity of the molecularly homogeneous PFC→PAG type against two heterogeneous classes in several two-alternative choice tasks in freely-moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics.

Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFC→PAG (periaqueductal gray). By comparing Ca(2+) activity of the molecularly homogeneous PFC→PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics.

Drugs of abuse elicit powerful experiences that engage populations of neurons broadly distributed throughout the brain. To determine how synaptic connectivity is organized to enable robust communication between populations of drug-activated neurons, we developed a complementary targeting system for monosynaptic rabies virus (RV) tracing that identifies direct inputs to activated versus non-activated neuronal populations. Analysis of over 100,000 synaptic input neurons demonstrated that cocaine-activated neurons comprise selectively connected, but broadly distributed, corticostriatal networks. Electrophysiological assays using optogenetics to stimulate activated versus non-activated inputs revealed stronger synapses between co-activated cortical pyramidal neurons and neurons in the dorsal striatum (DS). Repeated cocaine exposure further enhanced the connectivity specifically between drug-activated neurons in the orbitofrontal cortex (OFC) and co-active DS neurons. Selective chemogenetic silencing of cocaine-activated OFC neurons or their terminals in DS disrupted behavioral sensitization, demonstrating the utility of this methodology for identifying novel circuit elements that contribute to behavioral plasticity.

Serotonin neurons of the dorsal and median raphe nuclei (DR, MR) collectively innervate the entire forebrain and midbrain, modulating diverse physiology and behavior. To gain a fundamental understanding of their molecular heterogeneity, we used plate-based single-cell RNA-sequencing to generate a comprehensive dataset comprising eleven transcriptomically distinct serotonin neuron clusters. Systematic in situ hybridization mapped specific clusters to the principal DR, caudal DR, or MR. These transcriptomic clusters differentially express a rich repertoire of neuropeptides, receptors, ion channels, and transcription factors. We generated novel intersectional viral-genetic tools to access specific subpopulations. Whole-brain axonal projection mapping revealed that DR serotonin neurons co-expressing vesicular glutamate transporter-3 preferentially innervate the cortex, whereas those co-expressing thyrotropin-releasing hormone innervate subcortical regions in particular the hypothalamus. Reconstruction of 50 individual DR serotonin neurons revealed diverse and segregated axonal projection patterns at the single-cell level. Together, these results provide a molecular foundation of the heterogenous serotonin neuronal phenotypes.

The projection targets of a neuronal population are a key feature of its anatomical characterization. Historically, tissue sectioning, confocal microscopy, and manual scoring of specific regions of interest have been used to generate coarse summaries of mesoscale projectomes. We present here TrailMap, a 3D convolutional network for extracting axonal projections from intact cleared mouse brains imaged by light-sheet microscopy. TrailMap allows region-based quantification of total axon content in large and complex 3D structures after registration to a standard reference atlas. The identification of axonal structures as thin as one voxel benefits from data augmentation but also requires a loss function that tolerates errors in annotation. A network trained with volumes of serotonergic axons in all major brain regions can be generalized to map and quantify axons from thalamocortical, deep cerebellar, and cortical projection neurons, validating transfer learning as a tool to adapt the model to novel categories of axonal morphology. Speed of training, ease of use, and accuracy improve over existing tools without a need for specialized computing hardware. Given the recent emphasis on genetically and functionally defining cell types in neural circuit analysis, TrailMap will facilitate automated extraction and quantification of axons from these specific cell types at the scale of the entire mouse brain, an essential component of deciphering their connectivity.

Parenting is essential for the survival and wellbeing of mammalian offspring. However, we lack a circuit-level understanding of how distinct components of this behaviour are coordinated. Here we investigate how galanin-expressing neurons in the medial preoptic area (MPOAGal) of the hypothalamus coordinate motor, motivational, hormonal and social aspects of parenting in mice. These neurons integrate inputs from a large number of brain areas and the activation of these inputs depends on the animal’s sex and reproductive state. Subsets of MPOAGal neurons form discrete pools that are defined by their projection sites. While the MPOAGal population is active during all episodes of parental behaviour, individual pools are tuned to characteristic aspects of parenting. Optogenetic manipulation of MPOAGal projections mirrors this specificity, affecting discrete parenting components. This functional organization, reminiscent of the control of motor sequences by pools of spinal cord neurons, provides a new model for how discrete elements of a social behaviour are generated at the circuit level.

Brain functions rely on specific patterns of connectivity. Teneurins are evolutionarily conserved transmembrane proteins that instruct synaptic partner matching in Drosophila and are required for vertebrate visual system development. The roles of vertebrate Teneurins in connectivity beyond the visual system remain largely unknown and their mechanisms of action have not been demonstrated. Here we show that mouse Teneurin-3 is expressed in multiple topographically interconnected areas of the hippocampal region, including proximal CA1, distal subiculum, and medial entorhinal cortex. Viral-genetic analyses reveal that Teneurin-3 is required in both CA1 and subicular neurons for the precise targeting of proximal CA1 axons to distal subiculum. Furthermore, Teneurin-3 promotes homophilic adhesion in vitro in a splicing isoform-dependent manner. These findings demonstrate striking genetic heterogeneity across multiple hippocampal areas and suggest that Teneurin-3 may orchestrate the assembly of a complex distributed circuit in the mammalian brain via matching expression and homophilic attraction.

In motor neuroscience, state changes are hypothesized to time-lock neural assemblies coordinating complex movements, but evidence for this remains slender. We tested whether a discrete change from more autonomous to coherent spiking underlies skilled movement by imaging cerebellar Purkinje neuron complex spikes in mice making targeted forelimb-reaches. As mice learned the task, millimeter-scale spatiotemporally coherent spiking emerged ipsilateral to the reaching forelimb, and consistent neural synchronization became predictive of kinematic stereotypy. Before reach onset, spiking switched from more disordered to internally time-locked concerted spiking and silence. Optogenetic manipulations of cerebellar feedback to the inferior olive bi-directionally modulated neural synchronization and reaching direction. A simple model explained the reorganization of spiking during reaching as reflecting a discrete bifurcation in olivary network dynamics. These findings argue that to prepare learned movements, olivo-cerebellar circuits enter a self-regulated, synchronized state promoting motor coordination. State changes facilitating behavioral transitions may generalize across neural systems.

Water deprivation produces a drive to seek and consume water. How neural activity creates this motivation remains poorly understood. We used activity-dependent genetic labeling to characterize neurons activated by water deprivation in the hypothalamic median preoptic nucleus (MnPO). Single-cell transcriptional profiling revealed that dehydration-activated MnPO neurons consist of a single excitatory cell type. After optogenetic activation of these neurons, mice drank water and performed an operant lever-pressing task for water reward with rates that scaled with stimulation frequency. This stimulation was aversive, and instrumentally pausing stimulation could reinforce lever-pressing. Activity of these neurons gradually decreased over the course of an operant session. Thus, the activity of dehydration-activated MnPO neurons establishes a scalable, persistent, and aversive internal state that dynamically controls thirst-motivated behavior.

Identification of neural circuit changes that contribute to behavioural plasticity has routinely been conducted on candidate circuits that were preselected on the basis of previous results. Here we present an unbiased method for identifying experience-triggered circuit-level changes in neuronal ensembles in mice. Using rabies virus monosynaptic tracing, we mapped cocaine-induced global changes in inputs onto neurons in the ventral tegmental area. Cocaine increased rabies-labelled inputs from the globus pallidus externus (GPe), a basal ganglia nucleus not previously known to participate in behavioural plasticity triggered by drugs of abuse. We demonstrated that cocaine increased GPe neuron activity, which accounted for the increase in GPe labelling. Inhibition of GPe activity revealed that it contributes to two forms of cocaine-triggered behavioural plasticity, at least in part by disinhibiting dopamine neurons in the ventral tegmental area. These results suggest that rabies-based unbiased screening of changes in input populations can identify previously unappreciated circuit elements that critically support behavioural adaptations.

Slow, controlled breathing has been used for centuries to promote mental calming, and it is used clinically to suppress excessive arousal such as panic attacks. However, the physiological and neural basis of the relationship between breathing and higher-order brain activity is unknown. We found a neuronal subpopulation in the mouse preBotzinger complex (preBotC), the primary breathing rhythm generator, which regulates the balance between calm and arousal behaviors. Conditional, bilateral genetic ablation of the ~175 Cdh9/Dbx1 double-positive preBotC neurons in adult mice left breathing intact but increased calm behaviors and decreased time in aroused states. These neurons project to, synapse on, and positively regulate noradrenergic neurons in the locus coeruleus, a brain center implicated in attention, arousal, and panic that projects throughout the brain.

Haploinsufficiency of Retinoic Acid Induced 1 (RAI1) causes Smith-Magenis syndrome (SMS), which is associated with diverse neurodevelopmental and behavioral symptoms as well as obesity. RAI1 encodes a nuclear protein but little is known about its molecular function or the cell types responsible for SMS symptoms. Using genetically engineered mice, we found that Rai1 preferentially occupies DNA regions near active promoters and promotes the expression of a group of genes involved in circuit assembly and neuronal communication. Behavioral analyses demonstrated that pan-neural loss of Rai1 causes deficits in motor function, learning, and food intake. These SMS-like phenotypes are produced by loss of Rai1 function in distinct neuronal types: Rai1 loss in inhibitory neurons or subcortical glutamatergic neurons causes learning deficits, while Rai1 loss in Sim1+ or SF1+ cells causes obesity. By integrating molecular and organismal analyses, our study suggests potential therapeutic avenues for a complex neurodevelopmental disorder.

Although classically thought of as a motor circuit, the cerebellum is now understood to contribute to a wide variety of cognitive functions through its dense interconnections with the neocortex, the center of brain cognition. Recent investigations have shed light on the nature of cerebellar cognitive processing and information exchange with the neocortex. We review findings that demonstrate widespread reward-related cognitive input to the cerebellum, as well as new studies that have characterized the codependence of processing in the neocortex and cerebellum. Together, these data support a view of the neocortex–cerebellum circuit as a joint dynamic system both in classical sensorimotor contexts and reward-related, cognitive processing. These studies have also expanded classical theory on the computations performed by the cerebellar circuit.

Serotonin neurons of the dorsal and medial raphe nuclei (DR and MR) collectively innervate the entire forebrain and midbrain, modulating diverse physiology and behavior. To gain a fundamental understanding of their molecular heterogeneity, we used plate-based single-cell RNA-sequencing to generate a comprehensive dataset comprising eleven transcriptomically distinct serotonin neuron clusters. Systematic in situ hybridization mapped specific clusters to the principal DR, caudal DR, or MR. These transcriptomic clusters differentially express a rich repertoire of neuropeptides, receptors, ion channels, and transcription factors. We generated novel intersectional viral-genetic tools to access specific subpopulations. Whole-brain axonal projection mapping revealed that DR serotonin neurons co-expressing vesicular glutamate transporter-3 preferentially innervate the cortex, whereas those co-expressing thyrotropin-releasing hormone innervate subcortical regions in particular the hypothalamus. Reconstruction of 50 individual DR serotonin neurons revealed segregated axonal projection patterns at the single-cell level. Together, these results provide a molecular foundation of the heterogenous serotonin neuronal phenotypes.

Plexins exhibit multitudinous, evolutionarily conserved functions in neural development. How Plexins employ their diverse structural motifs in vivo to perform distinct roles is unclear. We previously reported that Plexin B (PlexB) controls multiple steps during the assembly of the Drosophila olfactory circuit (Li et al., 2018b). Here, we systematically mutagenized structural motifs of PlexB and examined the function of these variants in these multiple steps: axon fasciculation, trajectory choice, and synaptic partner selection. We found that the extracellular Sema domain is essential for all three steps, the catalytic site of the intracellular RapGAP is engaged in none, and the intracellular GTPase-binding motifs are essential for trajectory choice and synaptic partner selection, but are dispensable for fasciculation. Moreover, extracellular PlexB cleavage serves as a regulatory mechanism of PlexB signaling. Thus, the divergent roles of PlexB motifs in distinct steps of neural development contribute to its functional versatility in neural circuit assembly.

Our understanding of the mechanisms of neural circuit assembly is far from complete. Identification of new wiring molecules with novel mechanisms of action will provide new insights into how complex and heterogeneous neural circuits assemble during development. Here, we performed an RNAi screen for cell-surface molecules and identified the leucine-rich-repeat containing transmembrane protein, Fish-lips (Fili), as a novel wiring molecule in the assembly of theDrosophilaolfactory circuit. Fili contributes to the precise targeting of both olfactory receptor neuron (ORN) axons as well as projection neuron (PN) dendrites. Cell-type-specific expression and genetic analyses suggest that Fili sends a trans-synaptic repulsive signal to neurites of non-partner classes that prevent their targeting to inappropriate glomeruli in the antennal lobe.Significance StatementIn the fruit fly olfactory system, 50 classes of olfactory receptor neurons (ORNs) make precise synaptic connections with 50 classes of corresponding projection neurons (PNs). Identification of wiring molecules in this circuit can provide insight into understanding neural circuit assembly. This paper reports the role of a transmembrane protein, Fish-lips (Fili), in forming specific connections in this circuit. We found that some ORN axons are repelled by Fili, which is present on dendrites of non-matching PN class, preventing them from targeting inappropriate glomeruli. Similarly, some PN dendrites are repelled by Fili expressed by non-matching ORN class for their correct targeting. Together, these results suggest that Fili mediates repulsion between axons and dendrites of non-synaptic partners to ensure precise wiring patterns.

The ultimate function of a neuron is determined by both its physiology and connectivity, but the transcriptional regulatory mechanisms that coordinate these two features are not well understood1–4. TheDrosophilaOlfactory receptor neurons (ORNs) provide an excellent system to investigate this question. As in mammals5, eachDrosophilaORN class is defined by the expression of a single olfactory receptor or a unique combination thereof, which determines their odor responses, and by the single glomerulus to which their axons target, which determines how sensory signals are represented in the brain6–10. In mammals, the coordination of olfactory receptor expression and wiring specificity is accomplished in part by olfactory receptors themselves regulating ORN wiring specificity11–13. However,Drosophilaolfactory receptors do not instruct axon targeting6, 14, raising the question as to how receptor expression and wiring specificity are coordinated. Using single-cell RNA-sequencing and genetic analysis, we identified 33 transcriptomic clusters for fly ORNs. We unambiguously mapped 17 to glomerular classes, demonstrating that transcriptomic clusters correspond well with anatomically and physiologically defined ORN classes. We found that each ORN expresses ~150 transcription factors (TFs), and identified a master TF that regulates both olfactory receptor expression and wiring specificity. A second TF plays distinct roles, regulating only receptor expression in one class and only wiring in another. Thus, fly ORNs utilize diverse transcriptional strategies to coordinate physiology and connectivity.

Cortical networks are characterized by sparse connectivity, with synapses found at only a subset of axo-dendritic contacts. Yet within these networks, neurons can exhibit high connection probabilities, suggesting that cell-intrinsic factors, not proximity, determine connectivity. Here, we identify ephrin-B3 (eB3) as a factor that determines synapse density by mediating a cell-cell competition that requires ephrin-B-EphB signaling. In a microisland culture system designed to isolate cell-cell competition, we find that eB3 determines winning and losing neurons in a contest for synapses. In a Mosaic Analysis with Double Markers (MADM) genetic mouse model system in vivo the relative levels of eB3 control spine density in layer 5 and 6 neurons. MADM cortical neurons in vitro reveal that eB3 controls synapse density independently of action potential-driven activity. Our findings illustrate a new class of competitive mechanism mediated by trans-synaptic organizing proteins which control the number of synapses neurons receive relative to neighboring neurons.