DYRK1A, a ubiquitously expressed kinase, is linked to the dominant intellectual developmental disorder, microcephaly, and Down syndrome in humans. It regulates numerous cellular processes such as cell cycle, vesicle trafficking, and microtubule assembly. DYRK1A is a critical regulator of organ growth; however, how it regulates organ growth is not fully understood. Here, we show that the knockdown of DYRK1A in mammalian cells results in reduced cell size, which depends on mTORC1. Using proteomic approaches, we found that DYRK1A interacts with the tuberous sclerosis complex (TSC) proteins, namely TSC1 and TSC2, which negatively regulate mTORC1 activation. Furthermore, we show that DYRK1A phosphorylates TSC2 at T1462, a modification known to inhibit TSC activity and promote mTORC1 activity. We also found that the reduced cell growth upon knockdown of DYRK1A can be rescued by overexpression of RHEB, an activator of mTORC1. Our findings suggest that DYRK1A inhibits TSC complex activity through inhibitory phosphorylation on TSC2, thereby promoting mTORC1 activity. Furthermore, using the Drosophila neuromuscular junction as a model, we show that the mnb, the fly homologs of DYRK1A, is rescued by RHEB overexpression, suggesting a conserved role of DYRK1A in TORC1 regulation.

LncRNAs are involved in modulating the individual risk and the severity of progression in metabolic dysfunction-associated fatty liver disease (MASLD), but their precise roles remain largely unknown. This study aimed to investigate the role of lncRNA Snhg3 in the development and progression of MASLD, along with the underlying mechanisms. The result showed that Snhg3 was significantly downregulated in the liver of high-fat diet-induced obesity (DIO) mice. Notably, palmitic acid promoted the expression of Snhg3 and overexpression of Snhg3 increased lipid accumulation in primary hepatocytes. Furthermore, hepatocyte-specific Snhg3 deficiency decreased body and liver weight, alleviated hepatic steatosis and promoted hepatic fatty acid metabolism in DIO mice, whereas overexpression induced the opposite effect. Mechanistically, Snhg3 promoted the expression, stability and nuclear localization of SND1 protein via interacting with SND1, thereby inducing K63-linked ubiquitination modification of SND1. Moreover, Snhg3 decreased the H3K27me3 level and induced SND1-mediated chromatin loose remodeling, thus reducing H3K27me3 enrichment at the Pparg promoter and enhancing PPARγ expression. The administration of PPARγ antagonist T0070907 improved Snhg3-aggravated hepatic steatosis. Our study revealed a new signaling pathway, Snhg3/SND1/H3K27me3/PPARγ, responsible for mice MASLD and indicates that lncRNA-mediated epigenetic modification has a crucial role in the pathology of MASLD.

During both sleep and awake immobility, hippocampal place cells reactivate time-compressed versions of sequences representing recently experienced trajectories in a phenomenon known as replay. Intriguingly, spontaneous sequences can also correspond to forthcoming trajectories in novel environments experienced later, in a phenomenon known as preplay. Here, we present a model showing that sequences of spikes correlated with the place fields underlying spatial trajectories in both previously experienced and future novel environments can arise spontaneously in neural circuits with random, clustered connectivity rather than pre-configured spatial maps. Moreover, the realistic place fields themselves arise in the circuit from minimal, landmark-based inputs. We find that preplay quality depends on the network's balance of cluster isolation and overlap, with optimal preplay occurring in small-world regimes of high clustering yet short path lengths. We validate the results of our model by applying the same place field and preplay analyses to previously published rat hippocampal place cell data. Our results show that clustered recurrent connectivity can generate spontaneous preplay and immediate replay of novel environments. These findings support a framework whereby novel sensory experiences become associated with preexisting "pluripotent" internal neural activity patterns.

The male-limited inheritance of Y chromosomes favors alleles that increase male fitness, often at the expense of female fitness. Determining the mechanisms underlying these sexually antagonistic effects is challenging because it can require studying Y-linked alleles while they still segregate as polymorphisms. We used a Y chromosome polymorphism in the house fly, Musca domestica, to address this challenge. Two male determining Y chromosomes (YM and IIIM) segregate as stable polymorphisms in natural populations, and they differentially affect multiple traits, including male courtship performance. We identified differentially expressed genes encoding odorant binding proteins (in the Obp56h family) as candidate agents for the courtship differences. Through network analysis and allele-specific expression measurements, we identified multiple genes on the house fly IIIM chromosome that could serve as trans regulators of Obp56h gene expression. One of those genes is homologous to Drosophila melanogaster CG2120, which encodes a transcription factor that binds near Obp56h. Upregulation of CG2120 in D. melanogaster nervous tissues reduces copulation latency, consistent with this transcription factor acting as a negative regulator of Obp56h expression. The transcription factor gene, which we name speed date, demonstrates a molecular mechanism by which a Y-linked gene can evolve male-beneficial effects.

Most of Earth's biomass is composed of polysaccharides. During biomass decomposition, polysaccharides are degraded by heterotrophic bacteria as a nutrient and energy source and are thereby partly remineralized into CO2. As polysaccharides are heterogeneously distributed in nature, following the colonization and degradation of a polysaccharide hotspot the cells need to reach new polysaccharide hotspots. Even though many studies indicate that these degradation-dispersal cycles contribute to the carbon flow in marine systems, we know little about how cells alternate between polysaccharide degradation and motility, and which environmental factors trigger this behavioral switch. Here, we studied the growth of the marine bacterium Vibrio cyclitrophicus ZF270 on the abundant marine polysaccharide alginate, both in its soluble polymeric form as well as on its breakdown products. We used microfluidics coupled to time-lapse microscopy to analyze motility and growth of individual cells, and RNA sequencing to study associated changes in gene expression. We found that single cells grow at reduced rate on alginate until they form large groups that cooperatively break down the polymer. Exposing cell groups to digested alginate accelerates cell growth and changes the expression of genes involved in alginate degradation and catabolism, central metabolism, ribosomal biosynthesis, and transport. However, exposure to digested alginate also triggers cells to become motile and disperse from cell groups, proportionally increasing with the group size before the nutrient switch, and this is accompanied by high expression of genes involved in flagellar assembly, chemotaxis, and quorum sensing. The motile cells chemotax toward polymeric but not digested alginate, likely enabling them to find new polysaccharide hotspots. Overall, our findings reveal cellular mechanisms that might also underlie bacterial degradation-dispersal cycles, which influence the remineralization of biomass in marine environments.

Neuropeptides and neurotrophins are stored in and released from dense core vesicles (DCVs). While DCVs and synaptic vesicles (SVs) share fundamental SNARE/SM proteins for exocytosis, a detailed understanding of DCV exocytosis remains elusive. We recently identified the RAB3-RIM1 pathway to be essential for DCV, but not SV exocytosis, highlighting a significant distinction between the SV and DCV secretory pathways. Whether RIM1 is the only RAB3 effector that is essential for DCV exocytosis is currently unknown. In this study, we show that rabphilin-3A (RPH3A), a known downstream effector of RAB3A, is a negative regulator of DCV exocytosis. Using live-cell imaging at single-vesicle resolution with RPH3A deficient hippocampal mouse neurons, we show that DCV exocytosis increased threefold in the absence of RPH3A. RAB3A-binding deficient RPH3A lost its punctate distribution, but still restored DCV exocytosis to WT levels when re-expressed. SNAP25-binding deficient RPH3A did not rescue DCV exocytosis. In addition, we show that RPH3A did not travel with DCVs, but remained stationary at presynapses. RPH3A null neurons also had longer neurites, which was partly restored when ablating all regulated secretion with tetanus neurotoxin. Taken together, these results show that RPH3A negatively regulates DCV exocytosis, potentially also affecting neuron size. Furthermore, RAB3A interaction is required for the synaptic enrichment of RPH3A, but not for limiting DCV exocytosis. Instead, the interaction of RPH3A with SNAP25 is relevant for inhibiting DCV exocytosis.

Animals have the innate ability to select optimal defensive behaviors with appropriate intensity within specific contexts. The vomeronasal organ (VNO) serves as a primary sensory channel for detecting predator cues by relaying signals to the medial hypothalamic nuclei, particularly the ventromedial hypothalamus (VMH), which directly controls defensive behavioral outputs. Here, we demonstrate that cat saliva contains predator cues that signal the imminence of predator threat and modulate the intensity of freezing behavior through the VNO in mice. Cat saliva activates VNO neurons expressing the V2R-A4 subfamily of sensory receptors, and the number of VNO neurons activated in response to saliva correlates with both the freshness of saliva and the intensity of freezing behavior. Moreover, the number of VMH neurons activated by fresh, but not old, saliva positively correlates with the intensity of freezing behavior. Detailed analyses of the spatial distribution of activated neurons, as well as their overlap within the same individual mice, revealed that fresh and old saliva predominantly activate distinct neuronal populations within the VMH. Collectively, this study suggests that there is an accessory olfactory circuit in mice that is specifically tuned to time-sensitive components of cat saliva, which optimizes their defensive behavior to maximize their chance of survival according to the imminence of threat.

Several areas of the world suffer a notably high incidence of Shiga toxin-producing Escherichia coli. To assess the impact of persistent cross-species transmission systems on the epidemiology of E. coli O157:H7 in Alberta, Canada, we sequenced and assembled E. coli O157:H7 isolates originating from collocated cattle and human populations, 2007-2015. We constructed a timed phylogeny using BEAST2 using a structured coalescent model. We then extended the tree with human isolates through 2019 to assess the long-term disease impact of locally persistent lineages. During 2007-2015, we estimated that 88.5% of human lineages arose from cattle lineages. We identified 11 persistent lineages local to Alberta, which were associated with 38.0% (95% CI 29.3%, 47.3%) of human isolates. During the later period, six locally persistent lineages continued to be associated with human illness, including 74.7% (95% CI 68.3%, 80.3%) of reported cases in 2018 and 2019. Our study identified multiple locally evolving lineages transmitted between cattle and humans persistently associated with E. coli O157:H7 illnesses for up to 13 y. Locally persistent lineages may be a principal cause of the high incidence of E. coli O157:H7 in locations such as Alberta and provide opportunities for focused control efforts.

It has been well documented that cold is an enhancer of lipid metabolism in peripheral tissues, yet its effect on central nervous system lipid dynamics is underexplored. It is well recognized that cold acclimations enhance adipocyte functions, including white adipose tissue lipid lipolysis and beiging, and brown adipose tissue thermogenesis in mammals. However, it remains unclear whether and how lipid metabolism in the brain is also under the control of ambient temperature. Here, we show that cold exposure predominantly increases the expressions of the lipid lipolysis genes and proteins within the paraventricular nucleus of the hypothalamus (PVH) in male mice. Mechanistically, by using innovatively combined brain-region selective pharmacology and in vivo time-lapse photometry monitoring of lipid metabolism, we find that cold activates cells within the PVH and pharmacological inactivation of cells blunts cold-induced effects on lipid peroxidation, accumulation of lipid droplets, and lipid lipolysis in the PVH. Together, these findings suggest that PVH lipid metabolism is cold sensitive and integral to cold-induced broader regulatory responses.

Sensory experience during developmental critical periods has lifelong consequences for circuit function and behavior, but the molecular and cellular mechanisms through which experience causes these changes are not well understood. The Drosophila antennal lobe houses synapses between olfactory sensory neurons (OSNs) and downstream projection neurons (PNs) in stereotyped glomeruli. Many glomeruli exhibit structural plasticity in response to early-life odor exposure, indicating a general sensitivity of the fly olfactory circuitry to early sensory experience. We recently found that glia shape antennal lobe development in young adults, leading us to ask if glia also drive experience-dependent plasticity during this period. Here, we define a critical period for structural and functional plasticity of OSN-PN synapses in the ethyl butyrate (EB)-sensitive glomerulus VM7. EB exposure for the first 2 days post-eclosion drives large-scale reductions in glomerular volume, presynapse number, and post- synaptic activity. Crucially, pruning during the critical period has long-term consequences for circuit function since both OSN-PN synapse number and spontaneous activity of PNs remain persistently decreased following early-life odor exposure. The highly conserved engulfment receptor Draper is required for this critical period plasticity as ensheathing glia upregulate Draper, invade the VM7 glomerulus, and phagocytose OSN presynaptic terminals in response to critical-period EB exposure. Loss of Draper fully suppresses the morphological and physiological consequences of critical period odor exposure, arguing that phagocytic glia engulf intact synaptic terminals. These data demonstrate experience-dependent pruning of synapses and argue that Drosophila olfactory circuitry is a powerful model for defining the function of glia in critical period plasticity.

The amygdala is a subcortical region in the mesiotemporal lobe that plays a key role in emotional and sensory functions. Conventional neuroimaging experiments treat this structure as a single, uniform entity, but there is ample histological evidence for subregional heterogeneity in microstructure and function. The current study characterized subregional structure-function coupling in the human amygdala, integrating post-mortem histology and in vivo MRI at ultra-high fields. Core to our work was a novel neuroinformatics approach that leveraged multiscale texture analysis as well as non-linear dimensionality reduction techniques to identify salient dimensions of microstructural variation in a 3D post-mortem histological reconstruction of the human amygdala. We observed two axes of subregional variation in this region, describing inferior-superior as well as mediolateral trends in microstructural differentiation that in part recapitulated established atlases of amygdala subnuclei. Translating our approach to in vivo MRI data acquired at 7 Tesla, we could demonstrate the generalizability of these spatial trends across 10 healthy adults. We then cross-referenced microstructural axes with functional blood-oxygen-level dependent (BOLD) signal analysis obtained during task-free conditions, and revealed a close association of structural axes with macroscale functional network embedding, notably the temporo-limbic, default mode, and sensory-motor networks. Our novel multiscale approach consolidates descriptions of amygdala anatomy and function obtained from histological and in vivo imaging techniques.

N -Acetylneuraminic acid (Neu5Ac) is a negatively charged nine-carbon amino sugar that is often the peripheral sugar in human cell-surface glycoconjugates. Some bacteria scavenge, import, and metabolize Neu5Ac or redeploy it on their cell surfaces for immune evasion. The import of Neu5Ac by many bacteria is mediated by tripartite ATP-independent periplasmic (TRAP) transporters. We have previously reported the structures of SiaQM, a membrane-embedded component of the Haemophilus influenzae TRAP transport system, (Currie et al., 2024). However, none of the published structures contain Neu5Ac bound to SiaQM. This information is critical for defining the transport mechanism and for further structure-activity relationship studies. Here, we report the structures of Fusobacterium nucleatum SiaQM with and without Neu5Ac. Both structures are in an inward (cytoplasmic side) facing conformation. The Neu5Ac-bound structure reveals the interactions of Neu5Ac with the transporter and its relationship with the Na+ binding sites. Two of the Na+-binding sites are similar to those described previously. We identify a third metal-binding site that is further away and buried in the elevator domain. Ser300 and Ser345 interact with the C1-carboxylate group of Neu5Ac. Proteoliposome-based transport assays showed that Ser300-Neu5Ac interaction is critical for transport, whereas Ser345 is dispensable. Neu5Ac primarily interacts with residues in the elevator domain of the protein, thereby supporting the elevator with an operator mechanism. The residues interacting with Neu5Ac are conserved, providing fundamental information required to design inhibitors against this class of proteins.

Progression through the G1 phase of the cell cycle is the most highly regulated step in cellular division. We employed a chemogenetic approach to discover novel cellular networks that regulate cell cycle progression. This approach uncovered functional clusters of genes that altered sensitivity of cells to inhibitors of the G1/S transition. Mutation of components of the Polycomb Repressor Complex 2 rescued proliferation inhibition caused by the CDK4/6 inhibitor palbociclib, but not to inhibitors of S phase or mitosis. In addition to its core catalytic subunits, mutation of the PRC2.1 accessory protein MTF2, but not the PRC2.2 protein JARID2, rendered cells resistant to palbociclib treatment. We found that PRC2.1 (MTF2), but not PRC2.2 (JARID2), was critical for promoting H3K27me3 deposition at CpG islands genome-wide and in promoters. This included the CpG islands in the promoter of the CDK4/6 cyclins CCND1 and CCND2, and loss of MTF2 lead to upregulation of both CCND1 and CCND2. Our results demonstrate a role for PRC2.1, but not PRC2.2, in antagonizing G1 progression in a diversity of cell linages, including chronic myeloid leukemia (CML), breast cancer, and immortalized cell lines.

Manifold-learning is particularly useful to resolve the complex cellular state space from single-cell RNA sequences. While current manifold-learning methods provide insights into cell fate by inferring graph-based trajectory at cell level, challenges remain to retrieve interpretable biology underlying the diverse cellular states. Here, we described MGPfactXMBD, a model-based manifold-learning framework and capable to factorize complex development trajectories into independent bifurcation processes of gene sets, and thus enables trajectory inference based on relevant features. MGPfactXMBD offers a more nuanced understanding of the biological processes underlying cellular trajectories with potential determinants. When bench-tested across 239 datasets, MGPfactXMBD showed advantages in major quantity-control metrics, such as branch division accuracy and trajectory topology, outperforming most established methods. In real datasets, MGPfactXMBD recovered the critical pathways and cell types in microglia development with experimentally valid regulons and markers. Furthermore, MGPfactXMBD discovered evolutionary trajectories of tumor-associated CD8+ T cells and yielded new subtypes of CD8+ T cells with gene expression signatures significantly predictive of the responses to immune checkpoint inhibitor in independent cohorts. In summary, MGPfactXMBD offers a manifold-learning framework in scRNA-seq data which enables feature selection for specific biological processes and contributing to advance our understanding of biological determination of cell fate.

When observing others' behaviors, we continuously integrate their movements with the corresponding sounds to enhance perception and develop adaptive responses. However, how the human brain integrates these complex audiovisual cues based on their natural temporal correspondence remains unclear. Using electroencephalogram (EEG), we demonstrated that rhythmic cortical activity tracked the hierarchical rhythmic structures in audiovisually congruent human walking movements and footstep sounds. Remarkably, the cortical tracking effects exhibit distinct multisensory integration modes at two temporal scales: an additive mode in a lower-order, narrower temporal integration window (step cycle) and a super-additive enhancement in a higher-order, broader temporal window (gait cycle). Furthermore, while neural responses at the lower-order timescale reflect a domain-general audiovisual integration process, cortical tracking at the higher-order timescale is exclusively engaged in the integration of biological motion cues. In addition, only this higher-order, domain-specific cortical tracking effect correlates with individuals' autistic traits, highlighting its potential as a neural marker for autism spectrum disorder. These findings unveil the multifaceted mechanism whereby rhythmic cortical activity supports the multisensory integration of human motion, shedding light on how neural coding of hierarchical temporal structures orchestrates the processing of complex, natural stimuli across multiple timescales.

The lateral line system enables fishes and aquatic-stage amphibians to detect local water movement via mechanosensory hair cells in neuromasts, and many species to detect weak electric fields via electroreceptors (modified hair cells) in ampullary organs. Both neuromasts and ampullary organs develop from lateral line placodes, but the molecular mechanisms underpinning ampullary organ formation are understudied relative to neuromasts. This is because the ancestral lineages of zebrafish (teleosts) and Xenopus (frogs) independently lost electroreception. We identified Bmp5 as a promising candidate via differential RNA-seq in an electroreceptive ray-finned fish, the Mississippi paddlefish (Polyodon spathula; Modrell et al., 2017, eLife 6: e24197). In an experimentally tractable relative, the sterlet sturgeon (Acipenser ruthenus), we found that Bmp5 and four other Bmp pathway genes are expressed in the developing lateral line, and that Bmp signalling is active. Furthermore, CRISPR/Cas9-mediated mutagenesis targeting Bmp5 in G0-injected sterlet embryos resulted in fewer ampullary organs. Conversely, when Bmp signalling was inhibited by DMH1 treatment shortly before the formation of ampullary organ primordia, supernumerary ampullary organs developed. These data suggest that Bmp5 promotes ampullary organ development, whereas Bmp signalling via another ligand(s) prevents their overproduction. Taken together, this demonstrates opposing roles for Bmp signalling during ampullary organ formation.

Wnt/β-catenin signaling directs animal development and tissue renewal in a tightly controlled, cell- and tissue-specific manner. In the mammalian central nervous system, the atypical ligand Norrin controls angiogenesis and maintenance of the blood-brain barrier and blood-retina barrier through the Wnt/β-catenin pathway. Like Wnt, Norrin activates signaling by binding and heterodimerizing the receptors Frizzled (Fzd) and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to membrane recruitment of the intracellular transducer Dishevelled (Dvl) and ultimately stabilizing the transcriptional coactivator β-catenin. Unlike Wnt, the cystine knot ligand Norrin only signals through Fzd4 and additionally requires the co-receptor Tetraspanin12 (Tspan12); however, the mechanism underlying Tspan12-mediated signal enhancement is unclear. It has been proposed that Tspan12 integrates into the Norrin-Fzd4 complex to enhance Norrin-Fzd4 affinity or otherwise allosterically modulate Fzd4 signaling. Here, we measure direct, high-affinity binding between purified Norrin and Tspan12 in a lipid environment and use AlphaFold models to interrogate this interaction interface. We find that Tspan12 and Fzd4 can simultaneously bind Norrin and that a pre-formed Tspan12/Fzd4 heterodimer, as well as cells co-expressing Tspan12 and Fzd4, more efficiently capture low concentrations of Norrin than Fzd4 alone. We also show that Tspan12 competes with both heparan sulfate proteoglycans and LRP6 for Norrin binding and that Tspan12 does not impact Fzd4-Dvl affinity in the presence or absence of Norrin. Our findings suggest that Tspan12 does not allosterically enhance Fzd4 binding to Norrin or Dvl, but instead functions to directly capture Norrin upstream of signaling.

The basal ganglia (BG) are an evolutionarily conserved and phylogenetically old set of sub-cortical nuclei that guide action selection, evaluation, and reinforcement. The entopeduncular nucleus (EP) is a major BG output nucleus that contains a population of GABA/glutamate cotransmitting neurons (EPSst+) that specifically target the lateral habenula (LHb) and whose function in behavior remains mysterious. Here, we use a probabilistic switching task that requires an animal to maintain flexible relationships between action selection and evaluation to examine when and how GABA/glutamate cotransmitting neurons contribute to behavior. We find that EPSst+ neurons are strongly engaged during this task and show bidirectional changes in activity during the choice and outcome periods of a trial. We then tested the effects of either permanently blocking cotransmission or modifying the GABA/glutamate ratio on behavior in well-trained animals. Neither manipulation produced detectable changes in behavior despite significant changes in synaptic transmission in the LHb, demonstrating that the outputs of these neurons are not required for ongoing action-outcome updating in a probabilistic switching task.

Different speeds of locomotion require heterogeneous spinal populations, but a common mode of rhythm generation is presumed to exist. Here, we explore the cellular versus synaptic origins of spinal rhythmicity at different speeds by performing electrophysiological recordings from premotor excitatory interneurons in larval zebrafish. Chx10-labeled V2a neurons are divided into at least two morphological subtypes proposed to play distinct roles in timing and intensity control. Consistent with distinct rhythm generating and output patterning functions within the spinal V2a population, we find that descending subtypes are recruited exclusively at slow or fast speeds and exhibit intrinsic cellular properties suitable for rhythmogenesis at those speeds, while bifurcating subtypes are recruited more reliably at all speeds and lack appropriate rhythmogenic cellular properties. Unexpectedly, however, phasic firing patterns during locomotion in rhythmogenic and non-rhythmogenic V2a neurons alike are best explained by distinct modes of synaptic inhibition linked to cell type and speed. At fast speeds reciprocal inhibition in descending V2a neurons supports phasic firing, while recurrent inhibition in bifurcating V2a neurons helps pattern motor output. In contrast, at slow speeds recurrent inhibition in descending V2a neurons supports phasic firing, while bifurcating V2a neurons rely on reciprocal inhibition alone to pattern output. Our findings suggest cell-type-specific, not common, modes of rhythmogenesis generate and coordinate different speeds of locomotion.

In the mammalian neocortex, inhibition is important for dynamically balancing excitation and shaping the response properties of cells and circuits. The various computational functions of inhibition are thought to be mediated by different inhibitory neuron types, of which a large diversity exists in several species. Current understanding of the function and connectivity of distinct inhibitory neuron types has mainly derived from studies in transgenic mice. However, it is unknown whether knowledge gained from mouse studies applies to the non-human primate, the model system closest to humans. The lack of viral tools to selectively access inhibitory neuron types has been a major impediment to studying their function in the primate. Here, we have thoroughly validated and characterized several recently developed viral vectors designed to restrict transgene expression to GABAergic cells or their parvalbumin (PV) subtype, and identified two types that show high specificity and efficiency in marmoset V1. We show that in marmoset V1, AAV-h56D induces transgene expression in GABAergic cells with up to 91-94% specificity and 79% efficiency, but this depends on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86-90% efficiency, depending on layer. Thus, these viral vectors are promising tools for studying GABA and PV cell function and connectivity in the primate cortex.