Ring-shaped odor coding in the antennal lobe of migratory locusts.

The representation of odors in the locust antennal lobe with its >2,000 glomeruli has long remained a perplexing puzzle. We employed the CRISPR-Cas9 system to generate transgenic locusts expressing the genetically encoded calcium indicator GCaMP in olfactory sensory neurons. Using two-photon functional imaging, we mapped the spatial activation patterns representing a wide range of ecologically relevant odors across all six developmental stages. Our findings reveal a functionally ring-shaped organization of the antennal lobe composed of specific glomerular clusters. This configuration establishes an odor-specific chemotopic representation by encoding different chemical classes and ecologically distinct odors in the form of glomerular rings. The ring-shaped glomerular arrangement, which we confirm by selective targeting of OR70a-expressing sensory neurons, occurs throughout development, and the odor-coding pattern within the glomerular population is consistent across developmental stages. Mechanistically, this unconventional spatial olfactory code reflects the locust-specific and multiplexed glomerular innervation pattern of the antennal lobe.

Sparse and stereotyped encoding implicates a core glomerulus for ant alarm behavior.

Ants communicate via large arrays of pheromones and possess expanded, highly complex olfactory systems, with antennal lobes in the brain comprising up to ∼500 glomeruli. This expansion implies that odors could activate hundreds of glomeruli, which would pose challenges for higher-order processing. To study this problem, we generated transgenic ants expressing the genetically encoded calcium indicator GCaMP in olfactory sensory neurons. Using two-photon imaging, we mapped complete glomerular responses to four ant alarm pheromones. Alarm pheromones robustly activated ≤6 glomeruli, and activity maps for the three pheromones inducing panic alarm in our study species converged on a single glomerulus. These results demonstrate that, rather than using broadly tuned combinatorial encoding, ants employ precise, narrowly tuned, and stereotyped representations of alarm pheromones. The identification of a central sensory hub glomerulus for alarm behavior suggests that a simple neural architecture is sufficient to translate pheromone perception into behavioral outputs.

Cellular bases of olfactory circuit assembly revealed by systematic time-lapse imaging.

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 30 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 exhibiting "exploring branches" consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral axons 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.

Development of Concurrent Retinotopic Maps in the Fly Motion Detection Circuit.

Understanding how complex brain wiring is produced during development is a daunting challenge. In Drosophila, information from 800 retinal ommatidia is processed in distinct brain neuropiles, each subdivided into 800 matching retinotopic columns. The lobula plate comprises four T4 and four T5 neuronal subtypes. T4 neurons respond to bright edge motion, whereas T5 neurons respond to dark edge motion. Each is tuned to motion in one of the four cardinal directions, effectively establishing eight concurrent retinotopic maps to support wide-field motion. We discovered a mode of neurogenesis where two sequential Notch-dependent divisions of either a horizontal or a vertical progenitor produce matching sets of two T4 and two T5 neurons retinotopically coincident with pairwise opposite direction selectivity. We show that retinotopy is an emergent characteristic of this neurogenic program and derives directly from neuronal birth order. Our work illustrates how simple developmental rules can implement complex neural organization.

Phenotypic Convergence: Distinct Transcription Factors Regulate Common Terminal Features.

Transcription factors regulate the molecular, morphological, and physiological characteristics of neurons and generate their impressive cell-type diversity. To gain insight into the general principles that govern how transcription factors regulate cell-type diversity, we used large-scale single-cell RNA sequencing to characterize the extensive cellular diversity in the Drosophila optic lobes. We sequenced 55,000 single cells and assigned them to 52 clusters. We validated and annotated many clusters using RNA sequencing of FACS-sorted single-cell types and cluster-specific genes. To identify transcription factors responsible for inducing specific terminal differentiation features, we generated a "random forest" model, and we showed that the transcription factors Apterous and Traffic-jam are required in many but not all cholinergic and glutamatergic neurons, respectively. In fact, the same terminal characters often can be regulated by different transcription factors in different cell types, arguing for extensive phenotypic convergence. Our data provide a deep understanding of the developmental and functional specification of a complex brain structure.

Control of Synaptic Connectivity by a Network of Drosophila IgSF Cell Surface Proteins.

We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity.

Allosteric transcription stimulation by RNA polymerase II super elongation complex.

The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and the subcomplex ELL2-EAF1, which stimulates RNA polymerase II (RNA Pol II) elongation. Here, we report the cryoelectron microscopy (cryo-EM) structure of ELL2-EAF1 bound to a RNA Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the RNA Pol II lobe domain, explaining how SEC delivers P-TEFb to RNA Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that the stimulation of RNA elongation requires the dimerization module and the ELL2 linker that tethers the module to the RNA Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a closed conformation of the RNA Pol II active center cleft.

Missense variants in ANO4 cause sporadic encephalopathic or familial epilepsy with evidence for a dominant-negative effect.

Anoctamins are a family of Ca2+-activated proteins that may act as ion channels and/or phospholipid scramblases with limited understanding of function and disease association. Here, we identified five de novo and two inherited missense variants in ANO4 (alias TMEM16D) as a cause of fever-sensitive developmental and epileptic or epileptic encephalopathy (DEE/EE) and generalized epilepsy with febrile seizures plus (GEFS+) or temporal lobe epilepsy. In silico modeling of the ANO4 structure predicted that all identified variants lead to destabilization of the ANO4 structure. Four variants are localized close to the Ca2+ binding sites of ANO4, suggesting impaired protein function. Variant mapping to the protein topology suggests a preliminary genotype-phenotype correlation. Moreover, the observation of a heterozygous ANO4 deletion in a healthy individual suggests a dysfunctional protein as disease mechanism rather than haploinsufficiency. To test this hypothesis, we examined mutant ANO4 functional properties in a heterologous expression system by patch-clamp recordings, immunocytochemistry, and surface expression of annexin A5 as a measure of phosphatidylserine scramblase activity. All ANO4 variants showed severe loss of ion channel function and DEE/EE associated variants presented mild loss of surface expression due to impaired plasma membrane trafficking. Increased levels of Ca2+-independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca2+-dependent scramblase activity were observed. Co-transfection with ANO4 wild-type suggested a dominant-negative effect. In summary, we expand the genetic base for both encephalopathic sporadic and inherited fever-sensitive epilepsies and link germline variants in ANO4 to a hereditary disease.

Serine hydroxymethyl transferase is required for optic lobe neuroepithelia development in Drosophila.

Cell fate and growth require one-carbon units for the biosynthesis of nucleotides, methylation reactions and redox homeostasis, provided by one-carbon metabolism. Consistently, defects in one-carbon metabolism lead to severe developmental defects, such as neural tube defects. However, the role of this pathway during brain development and in neural stem cell regulation is poorly understood. To better understand the role of one carbon metabolism we focused on the enzyme Serine hydroxymethyl transferase (Shmt), a key factor in the one-carbon cycle, during Drosophila brain development. We show that, although loss of Shmt does not cause obvious defects in the central brain, it leads to severe phenotypes in the optic lobe. The shmt mutants have smaller optic lobe neuroepithelia, partly justified by increased apoptosis. In addition, shmt mutant neuroepithelia have morphological defects, failing to form a lamina furrow, which likely explains the observed absence of lamina neurons. These findings show that one-carbon metabolism is crucial for the normal development of neuroepithelia, and consequently for the generation of neural progenitor cells and neurons. These results propose a mechanistic role for one-carbon during brain development.

Embryonic organizer specification in the mud snail Ilyanassa obsoleta depends on intercellular signaling.

In early embryos of the caenogastropod snail Ilyanassa obsoleta, cytoplasmic segregation of a polar lobe is required for establishment of the D quadrant founder cell, empowering its great-granddaughter macromere 3D to act as a single-celled organizer that induces ectodermal pattern along the secondary body axis of the embryo. We present evidence that polar lobe inheritance is not sufficient to specify 3D potential, but rather makes the D macromere lineage responsive to some intercellular signal(s) required for normal expression of 3D-specific phenotypes. Experimental removal of multiple micromeres resulted in loss of organizer-linked MAPK activation, complete and specific defects of organizer-dependent larval organs, and progressive cell cycle retardation, leading to equalization of the normally accelerated division schedule of 3D (relative to the third-order macromeres of the A, B and C quadrants). Ablation of the second-quartet micromere 2d greatly potentiated the effects of first micromere quartet ablation. Our findings link organizer activation in I. obsoleta to the putative ancestral spiralian mechanism in which a signal from micromeres leads to specification of 3D among four initially equivalent macromeres.

Embryonic organizer specification in the mud snail Ilyanassa obsoleta depends on intercellular signaling.

In early embryos of the caenogastropod snail Ilyanassa obsoleta, cytoplasmic segregation of a polar lobe is required for establishment of the D quadrant founder cell, empowering its great-granddaughter macromere 3D to act as a single-celled organizer that induces ectodermal pattern along the secondary body axis of the embryo. We present evidence that polar lobe inheritance is not sufficient to specify 3D potential, but rather makes the D macromere lineage responsive to some intercellular signal(s) required for normal expression of 3D-specific phenotypes. Experimental removal of multiple micromeres resulted in loss of organizer-linked MAPK activation, complete and specific defects of organizer-dependent larval organs, and progressive cell cycle retardation, leading to equalization of the normally accelerated division schedule of 3D (relative to the third-order macromeres of the A, B and C quadrants). Ablation of the second-quartet micromere 2d greatly potentiated the effects of first micromere quartet ablation. Our findings link organizer activation in I. obsoleta to the putative ancestral spiralian mechanism in which a signal from micromeres leads to specification of 3D among four initially equivalent macromeres.

A chromatin remodelling SWI/SNF subunit, Snr1, regulates neural stem cell determination and differentiation.

Coordinated spatio-temporal regulation of the determination and differentiation of neural stem cells is essential for brain development. Failure to integrate multiple factors leads to defective brain structures or tumour formation. Previous studies suggest changes of chromatin state are needed to direct neural stem cell differentiation, but the mechanisms are unclear. Analysis of Snr1, the Drosophila orthologue of SMARCB1, an ATP-dependent chromatin remodelling protein, identified a key role in regulating the transition of neuroepithelial cells into neural stem cells and subsequent differentiation of neural stem cells into the cells needed to build the brain. Loss of Snr1 in neuroepithelial cells leads to premature neural stem cell formation. Additionally, loss of Snr1 in neural stem cells results in inappropriate perdurance of neural stem cells into adulthood. Snr1 reduction in neuroepithelial or neural stem cells leads to the differential expression of target genes. We find that Snr1 is associated with the actively transcribed chromatin region of these target genes. Thus, Snr1 likely regulates the chromatin state in neuroepithelial cells and maintains chromatin state in neural stem cells for proper brain development.

Cellular remodeling and JAK inhibition promote zygotic gene expression in the Ciona germline.

Transcription control is a major determinant of cell fate decisions in somatic tissues. By contrast, early germline fate specification in numerous vertebrate and invertebrate species relies extensively on RNA-level regulation, exerted on asymmetrically inherited maternal supplies, with little-to-no zygotic transcription. However delayed, a maternal-to-zygotic transition is nevertheless poised to complete the deployment of pre-gametic programs in the germline. Here, we focus on early germline specification in the tunicate Ciona to study zygotic genome activation. We first demonstrate that a peculiar cellular remodeling event excludes localized postplasmic Pem-1 mRNA, which encodes the general inhibitor of transcription. Subsequently, zygotic transcription begins in Pem-1-negative primordial germ cells (PGCs), as revealed by histochemical detection of elongating RNA Polymerase II, and nascent Mef2 transcripts. In addition, we uncover a provisional antagonism between JAK and MEK/BMPRI/GSK3 signaling, which controls the onset of zygotic gene expression, following cellular remodeling of PGCs. We propose a 2-step model for the onset of zygotic transcription in the Ciona germline and discuss the significance of germ plasm dislocation and remodeling in the context of developmental fate specification.

AcrIIC4 inhibits type II-C Cas9 by preventing R-loop formation.

Anti-CRISPR (Acr) proteins are encoded by phages and other mobile genetic elements and inhibit host CRISPR-Cas immunity using versatile strategies. AcrIIC4 is a broad-spectrum Acr that inhibits the type II-C CRISPR-Cas9 system in several species by an unknown mechanism. Here, we determined a series of structures of Haemophilus parainfluenzae Cas9 (HpaCas9)-sgRNA in complex with AcrIIC4 and/or target DNA, as well as the crystal structure of AcrIIC4 alone. We found that AcrIIC4 resides in the crevice between the REC1 and REC2 domains of HpaCas9, where its extensive interactions restrict the mobility of the REC2 domain and prevent the unwinding of target double-stranded (ds) DNA at the PAM-distal end. Therefore, the full-length guide RNA:target DNA heteroduplex fails to form in the presence of AcrIIC4, preventing Cas9 nuclease activation. Altogether, our structural and biochemical studies illuminate a unique Acr mechanism that allows DNA binding to the Cas9 effector complex but blocks its cleavage by preventing R-loop formation, a key step supporting DNA cleavage by Cas9.

Chinese Verbal Fluency Deficiency in Temporal Lobe Epilepsy with and without Hippocampal Sclerosis: A Multiscale Study.

To test a Chinese character version of the phonemic verbal fluency task in patients with temporal lobe epilepsy (TLE) and assess the verbal fluency deficiency pattern in TLE with and without hippocampal sclerosis, a cross-sectional study was conducted including 30 patients with TLE and hippocampal sclerosis (TLE-HS), 28 patients with TLE and without hippocampal sclerosis (TLE-NHS), and 29 demographically matched healthy controls (HC). Both sexes were enrolled. Participants finished a Chinese character verbal fluency (VFC) task during functional MRI. The activation/deactivation maps, functional connectivity, degree centrality, and community features of the left frontal and temporal regions were compared. A neural network classification model was applied to differentiate TLE-HS and TLE-NHS using functional statistics. The VFC scores were correlated with semantic fluency in HC while correlated with phonemic fluency in TLE-NHS. Activation and deactivation deficiency was observed in TLE-HS and TLE-NHS (p < 0.001, k ≥ 10). Functional connectivity, degree centrality, and community features of anterior inferior temporal gyri were impaired in TLE-HS and retained or even enhanced in TLE-NHS (p < 0.05, FDR-corrected). The functional connectivity was correlated with phonemic fluency (p < 0.05, FDR-corrected). The neural network classification reached an area under the curve of 0.90 in diagnosing hippocampal sclerosis. The VFC task is a Chinese phonemic verbal fluency task suitable for clinical application in TLE. During the VFC task, functional connectivity of phonemic circuits was impaired in TLE-HS and was enhanced in TLE-NHS, representing a compensative phonemic searching strategy applied by patients with TLE-NHS.

Human Hippocampal Ripples Signal Encoding of Episodic Memories.

Direct human brain recordings have confirmed the presence of high-frequency oscillatory events, termed ripples, during awake behavior. While many prior studies have focused on medial temporal lobe (MTL) ripples during memory retrieval, here we investigate ripples during memory encoding. Specifically, we ask whether ripples during encoding predict whether and how memories are subsequently recalled. Detecting ripples from MTL electrodes implanted in 116 neurosurgical participants (n = 61 male) performing a verbal episodic memory task, we find that encoding ripples do not distinguish recalled from not recalled items in any MTL region, even as high-frequency activity during encoding predicts recall in these same regions. Instead, hippocampal ripples increase during encoding of items that subsequently lead to recall of temporally and semantically associated items during retrieval, a phenomenon known as clustering. This subsequent clustering effect arises specifically when hippocampal ripples co-occur during encoding and retrieval, suggesting that ripples mediate both encoding and reinstatement of episodic memories.

Encoding of Visual Objects in the Human Medial Temporal Lobe.

The human medial temporal lobe (MTL) plays a crucial role in recognizing visual objects, a key cognitive function that relies on the formation of semantic representations. Nonetheless, it remains unknown how visual information of general objects is translated into semantic representations in the MTL. Furthermore, the debate about whether the human MTL is involved in perception has endured for a long time. To address these questions, we investigated three distinct models of neural object coding-semantic coding, axis-based feature coding, and region-based feature coding-in each subregion of the human MTL, using high-resolution fMRI in two male and six female participants. Our findings revealed the presence of semantic coding throughout the MTL, with a higher prevalence observed in the parahippocampal cortex (PHC) and perirhinal cortex (PRC), while axis coding and region coding were primarily observed in the earlier regions of the MTL. Moreover, we demonstrated that voxels exhibiting axis coding supported the transition to region coding and contained information relevant to semantic coding. Together, by providing a detailed characterization of neural object coding schemes and offering a comprehensive summary of visual coding information for each MTL subregion, our results not only emphasize a clear role of the MTL in perceptual processing but also shed light on the translation of perception-driven representations of visual features into memory-driven representations of semantics along the MTL processing pathway.

Visual Circuits for Direction Selectivity.

Images projected onto the retina of an animal eye are rarely still. Instead, they usually contain motion signals originating either from moving objects or from retinal slip caused by self-motion. Accordingly, motion signals tell the animal in which direction a predator, prey, or the animal itself is moving. At the neural level, visual motion detection has been proposed to extract directional information by a delay-and-compare mechanism, representing a classic example of neural computation. Neurons responding selectively to motion in one but not in the other direction have been identified in many systems, most prominently in the mammalian retina and the fly optic lobe. Technological advances have now allowed researchers to characterize these neurons' upstream circuits in exquisite detail. Focusing on these upstream circuits, we review and compare recent progress in understanding the mechanisms that generate direction selectivity in the early visual system of mammals and flies.

Differential reorganization of episodic and semantic memory systems in epilepsy-related mesiotemporal pathology.

Declarative memory encompasses episodic and semantic divisions. Episodic memory captures singular events with specific spatiotemporal relationships, while semantic memory houses context-independent knowledge. Behavioural and functional neuroimaging studies have revealed common and distinct neural substrates of both memory systems, implicating mesiotemporal lobe (MTL) regions such as the hippocampus and distributed neocortices. Here, we explored declarative memory system reorganization in patients with unilateral temporal lobe epilepsy (TLE) as a human disease model to test the impact of variable degrees of MTL pathology on memory function. Our cohort included 31 patients with TLE as well as 60 age and sex-matched healthy controls, and all participants underwent episodic and semantic retrieval tasks during a multimodal MRI session. The functional MRI tasks were closely matched in terms of stimuli and trial design. Capitalizing on non-linear connectome gradient mapping techniques, we derived task-based functional topographies during episodic and semantic memory states, both in the MTL and in neocortical networks. Comparing neocortical and hippocampal functional gradients between TLE patients and healthy controls, we observed a marked topographic reorganization of both neocortical and MTL systems during episodic memory states. Neocortical alterations were characterized by reduced functional differentiation in TLE across lateral temporal and midline parietal cortices in both hemispheres. In the MTL, on the other hand, patients presented with a more marked functional differentiation of posterior and anterior hippocampal segments ipsilateral to the seizure focus and pathological core, indicating perturbed intrahippocampal connectivity. Semantic memory reorganization was also found in bilateral lateral temporal and ipsilateral angular regions, while hippocampal functional topographies were unaffected. Leveraging MRI proxies of MTL pathology, we furthermore observed alterations in hippocampal microstructure and morphology that were associated with TLE-related functional reorganization during episodic memory. Moreover, correlation analysis and statistical mediation models revealed that these functional alterations contributed to behavioural deficits in episodic, but again not semantic memory in patients. Altogether, our findings suggest that semantic processes rely on distributed neocortical networks, while episodic processes are supported by a network involving both the hippocampus and neocortex. Alterations of such networks can provide a compact signature of state-dependent reorganization in conditions associated with MTL damage, such as TLE.

Amidst an amygdala renaissance in Alzheimer's disease.

The amygdala was highlighted as an early site for neurofibrillary tau tangle pathology in Alzheimer's disease in the seminal 1991 article by Braak and Braak. This knowledge has, however, only received traction recently with advances in imaging and image analysis techniques. Here, we provide a cross-disciplinary overview of pathology and neuroimaging studies on the amygdala. These studies provide strong support for an early role of the amygdala in Alzheimer's disease and the utility of imaging biomarkers of the amygdala in detecting early changes and predicting decline in cognitive functions and neuropsychiatric symptoms in early stages. We summarize the animal literature on connectivity of the amygdala, demonstrating that amygdala nuclei that show the earliest and strongest accumulation of neurofibrillary tangle pathology are those that are connected to brain regions that also show early neurofibrillary tangle accumulation. Additionally, we propose an alternative pathway of neurofibrillary tangle spreading within the medial temporal lobe between the amygdala and the anterior hippocampus. The proposed existence of this pathway is strengthened by novel experimental data on human functional connectivity. Finally, we summarize the functional roles of the amygdala, highlighting the correspondence between neurofibrillary tangle accumulation and symptomatic profiles in Alzheimer's disease. In summary, these findings provide a new impetus for studying the amygdala in Alzheimer's disease and a unique perspective to guide further study on neurofibrillary tangle spreading and the occurrence of neuropsychiatric symptoms in Alzheimer's disease.