eLife

Neuroimaging based pain decoding faces two underappreciated challenges: between subject variability that prevents classifiers from generalizing across patients, and within session cross validation designs that inflate reported accuracy by conflating within person and between person variance. Here we address both using portable functional near infrared spectroscopy (fNIRS) during pharmacologically verified local nerve anesthesia. Twentyfive patients with clinically painful teeth underwent 36 channel bilateral fNIRS during percussion before ("Pre") and after ("Post") local nerve anesthesia. In 13 block-success patients, a paired Pre versus Post comparison with healthy tooth control identified three temporal hemodynamic response function (HRF) features (late slope, mean first derivative, and baseline normalized amplitude) whose analgesia interaction effects (d = 0.63 to 0.79) exceeded that of raw general linear model (GLM) amplitude (d = 0.56), with a significant difference-in-differences interaction (p = 0.011). Per-patient calibration with these features yielded leave one subject out (LOSO) AUC = 0.68 to 0.76 for nonlinear classifiers (permutation p = 0.002), with HbO-specific feature selection achieving the best performance (RF AUC = 0.760); a healthy tooth negative control was non-significant. End to end deep learning on raw time series (CNN LSTM AUC = 0.719) was competitive with feature based classifiers, while linear models did not reach significance. Critically, head to head comparison of within-session CV and LOSO on the same data revealed mean inflation of +0.13 AUC across all model types, including deep learning, demonstrating that high within session accuracy alone does not establish subject-independent validity. Exploratory analyses suggested complementary roles for oxyhemoglobin (HbO; within patient analgesia detection) and deoxyhemoglobin (HbR; cross patient information), and that trial to trial response variability may complement amplitude for cross patient pain detection. These results show that per patient calibration with temporal HRF features supports subject independent analgesic-state detection under strict LOSO evaluation, and that within-session validation (standard in the fNIRS pain- decoding literature) can substantially overestimate performance.

Morphogenetic processes during animal development are remarkably invariant (Duboule, 1994; Hall, 1997; Kalinka et al., 2010; Raff, 1996). This stability is established by the interaction between genetic determination of developmental progression and the constraints imposed by the surrounding embryonic environment (Busby and Steventon, 2021; Gilmour et al., 2017; Gorfinkiel and Martinez Arias, 2021). We discovered that the germ band extension process in Drosophila is rather variable: instead of extending straight towards the head, the germ band tends to twist to the side. Through a combination of experiments and theory, we demonstrated that Scab integrin-mediated attachment to the vitelline envelope stabilizes the germ band and supports its straight extension. Our quantification of germ band extension dynamics also revealed a consistent handedness to the twist of the germ band. We showed that this left-right asymmetry can be altered by manipulating the expression of Myo1D, the molecular determinant of chirality in Drosophila (Lebreton et al., 2018). Our data thus suggest that Myo1D expression causes the early gastrulating blastoderm epithelium to already exhibit inherent chirality and that the resulting destabilization of germ band extension is suppressed by Scab-mediated friction between the blastoderm and the vitelline envelope.

How neighboring sarcomeres redistribute timing while a cardiomyocyte continues to beat, and how that coordination shapes mean HSO amplitude in the segment-average trace, remain unresolved. We reanalyzed sarcomere-length recordings from five consecutive sarcomeres in each of seven living neonatal rat cardiomyocytes and represented each valid time point by the four neighboring-pair phase relations that define a 16-state local phase network. During warming-induced hyperthermal sarcomeric oscillations (HSOs), the fraction of time with trackable local phase relations increased from 0.298 before warming to 0.956 (paired Wilcoxon P = 0.0156), enabling direct analysis of local reconfiguration. Successive local states were almost always connected by Hamming-1 edges, meaning that only one neighboring-pair relation changed at a time (34/35, 97.1%, before warming; 216/230, 93.9%, during HSOs). HSOs also increased occupancy of anti-phase-rich states with three or more anti-phase neighboring pairs (0.254 to 0.509, P = 0.0156). In a complementary cycle-level analysis of the same HSO window, Yvalid, the HSO amplitude of the valid-sarcomere mean trace, was closely approximated by the product of mean local HSO amplitude (A) and weighted synchrony across valid sarcomeres (Rw; pooled r = 0.992, normalized mean squared error = 0.015, {beta}1 = 0.948, {beta}0 {approx} 0). To link the binary local-state description to this continuous synchrony term, we derived a simple state-based synchrony factor from the local phase patterns; cycles with higher values showed higher Rw (cell-adjusted {beta} = 0.197, P = 0.0165). Blocked cross-validation showed that the A x Rw model markedly outperformed an additive alternative (pooled normalized mean squared error 0.0138 vs 0.1006), whereas simple history terms changed error only marginally. HSOs therefore do not reflect unstructured local disorder. Rather, they are characterized by a constrained neighboring-sarcomere phase topology, and mean HSO amplitude in the same segment is largely captured by a cycle-level relation between local amplitude and synchrony.

Cholinergic efferent neurons modulate sensory signaling in the peripheral vestibular system, but the cellular mechanisms underlying this modulation remain incompletely understood. In mammalian vestibular organs, type II hair cells (HC-II) receive efferent input and express 910-containing nicotinic acetylcholine receptors (nAChRs) that activate SK potassium channels and produce rapid hyperpolarization. Here, we examined the functional role of mAChRs in mouse vestibular HC-II using whole cell patch clamp recordings in whole tissue preparations of crista ampularis (P13-P17, male and female mice). Activation of mAChRs with oxotremorine-M inhibited voltage dependent outward currents, with the largest effects at depolarized membrane potentials. Further experiments revealed that this effect was mediated by inhibition of large conductance potassium (BK) channels: the BK antagonist iberiotoxin mimicked and occluded the muscarinic effect and muscarinic suppression was absent in mice with BK channel mutations. In contrast, blockade of SK channels with apamin did not prevent the muscarinic effect, indicating that mAChR signaling specifically targets BK mediated currents. In current clamp recordings, mAChR activation enhanced depolarization during strong current injections, consistent with increased hair cell excitability when BK channels were suppressed. These findings identify a previously unrecognized muscarinic efferent pathway in vestibular hair cells and reveal complementary cholinergic mechanisms that suppress responses to weak stimuli while enhancing responses to strong stimulation, providing a cellular basis for dynamic gain control in the vestibular periphery. Significance statementVestibular efferent signaling shapes how head movements are encoded, but its cellular mechanisms are incompletely understood. While nicotinic acetylcholine receptors are known to reduce excitability of type II vestibular hair cells (HC-II) via small conductance (SK) channels, the role of muscarinic receptors has remained unclear. Here we show that muscarinic receptor activation selectively inhibits large conductance (BK) potassium channels in HC-II, enhancing excitability during strong depolarization. This muscarinic pathway is mechanistically distinct from nicotinic signaling and operates at a different voltage range. Together, these findings reveal a dual efferent control strategy that differentially regulates hair cell responses to slow versus fast head movements, providing new insight into how the vestibular system filters sensory input across dynamic ranges.

Neighboring sarcomeres in a cardiomyocyte need not move in perfect synchrony, but it is unclear whether their local timing differences during hyperthermal sarcomeric oscillations (HSOs) are random or organized. Using the same five-sarcomere recordings that previously revealed a constrained 16-state neighboring-pair topology and an amplitude-synchrony relation for the segment-mean rapid signal (Shintani, 2026), we reanalyzed each fast HSO cycle as one local coordination summary. A topology-based circular coordinate showed that the 16 discrete patterns lie on a continuous within-cell order: adjacent positions were enriched for Hamming-1 changes and cycle-to-cycle angular drift was slower than expected by chance. Because each cell has an arbitrary angular zero point and direction, we aligned the cell-wise circles using shared local states as landmarks. This improved same-state concentration across cells from 0.582 to 0.852 (P = 0.0013). The clearest biological translation of the aligned coordinate was mismatch placement along the observed five-sarcomere segment. Aligned angle predicted edge-biased mismatch placement (joint P = 1.15 x 10-6), whereas raw angle did not (P = 0.55). Beat timing was weaker and signed strain less robust. These findings support a mesoscale view in which local HSO nonuniformity is structured: neighboring sarcomeres share a rephasing order, and that order is most readably expressed by where the local mismatch pocket lies along the chain. Significance statementCardiac contraction must convert noisy local events into a stable beat. This study identifies a measurable intermediate-scale variable in living cardiomyocytes. Fast HSO cycles do not wander randomly among local coordination patterns. After cross-cell alignment, they occupy a shared rephasing compass, and the clearest biological readout of that compass is where a local mismatch pocket sits along the observed five-sarcomere segment. This gives a concrete way to describe how local nonuniformity can remain structured rather than merely disruptive. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=51 SRC="FIGDIR/small/714639v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1d0d6deorg.highwire.dtl.DTLVardef@1cab2c8org.highwire.dtl.DTLVardef@9f9cadorg.highwire.dtl.DTLVardef@e75263_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO Five consecutive sarcomere-length traces were condensed into one local coordination summary per fast HSO cycle. A topology-based circular coordinate revealed a within-cell order, and cross-cell alignment turned that order into a shared rephasing compass. The clearest biological translation of that compass was where the local mismatch pocket was positioned along the observed five-sarcomere segment. C_FIG

Crosstalk across two major receptor families involved in signal transduction, namely receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), have been observed at different levels of their signaling cascades. Using newly developed enhanced bystander bioluminescence resonance energy transfer (ebBRET)-based biosensors that monitor the recruitment of SH2 domains to activated RTKs, we assessed the ability of GPCRs to modulate cellular localization of SH2 domains. Receptor-mediated activation of either Gq/11 or G12/13 but not Gs or Gi/o (e.g., thromboxane A2 receptor, TP, and type-2 protease activated receptor, PAR2) resulted in the plasma membrane (PM) dissociation of SH2 domains derived from RTKs effectors such as GRB2, STAT5 and PLC{gamma}1. The role of Gq/11, G12/13, Rho and downstream kinases in the subcellular SH2 domain redistribution was further confirmed using both pharmacological and genetic approaches. BRET imaging and spectrometric analyses showed that the dissociation of SH2 domains from the PM was accompanied by their accumulation in the nucleus and a reduction in RTK signaling activity, as determined using a STAT5 transcriptional assay. The effect of Gq/11 and G12/13 activation on STAT5 transcriptional activity was observed both in engineered systems and in HeLa cells endogenously expressing all the components of the regulatory mechanism. The Gq/11 / G12/13-mediated redistribution of SH2 domain-containing proteins represents an undescribed mechanism through which GPCRs regulate RTKs activity. Significance StatementThis study reveals a novel crosstalk mechanism between G protein coupled receptors and receptor tyrosine kinases showing that Gq/11 and G12/13 activation triggers Rho-dependent translocation of SH2-containing effector proteins, such as GRB2, PLC{gamma}1 and STAT5. This process causes compartmentalization inside the nucleus and thus reduces their availability at the plasma membrane, leading to attenuated RTK responses.

Allostery--regulation of a proteins activity by binding to an effector--is an essential functional feature of many proteins. Its structural basis is complex: a protein must bind effector at one site--an interaction that typically involves many complementary residues--and this binding event must change the proteins conformation at the distant active site. How allosteric proteins evolved this architecture is unknown because there are no cases in which the historical mechanisms by which allostery was acquired from nonallosteric precursors have been identified. Vertebrate hemoglobin (Hb) is a tetrameric protein whose oxygen affinity is reduced by binding organic phosphates. We used ancestral protein reconstruction, biochemical experiments, and in silico studies of protein structure and dynamics to identify the changes in protein sequence and structure that caused allosteric Hb to evolve from its non-allosteric dimeric precursor. We found that just two historical substitutions were sufficient to confer positive allostery on the ancestor--one that caused the dimer to tetramerize, and another that created an effector binding site in the tetramers central cavity. Two additional substitutions changed the effectors position and conferred the modern form of negative allostery. This short evolutionary path to allostery was possible because most of the key requirements for allostery already existed as by-products of the proteins structure: the tertiary transition between oxygenated and deoxygenated conformations is an ancient and intrinsic property of the globin fold. It affects the surface patch that ultimately mediated tetramer assembly, so as soon as tetramerization evolved because of one substitution on that surface, tertiary heterogeneity propagated to large-scale quaternary heterogeneity. The other substitution(s) conferred effector binding at a site within the tetramer that is preferentially accessible in the deoxygenated state. One of the most complex and essential protein phenomena therefore evolved via simple and intelligible mechanisms.

The morphology of the palate has long constituted the primary basis for differentiating between the two deepest clades of crown group birds, Neognathae and Palaeognathae. However, published literature on the bird palate is dominated by classical anatomical descriptions pre-dating the advent of contemporary three-dimensional imaging techniques, hindering our understanding of bird palate disparity and development. Pterygoid segmentation, the process by which the rostral portion of the pterygoid separates and fuses with the palatine during ontogeny in neognathous birds, remains a poorly understood aspect of avian cranial development despite giving rise to an important component of the cranial kinetic system. Here, we use micro-computed tomography to explore ontogenetic change of the palate during the process of pterygoid segmentation across an unprecedentedly broad taxonomic sample of immature and mature birds. We found that direct evidence of post-hatching pterygoid segmentation was restricted to the major avian subclade Neoaves. Based on morphological and topological similarities, we hypothesise that the rostral projection of the pterygoid observed in Anatidae/Anseres and potentially Anhimidae and Megapodiidae, which we term the hemipterygoid process, is homologous with the hemipterygoid of neoavians, though it does not undergo segmentation. We posit that the origin of a discrete hemipterygoid (as observable in some crownward stem-birds) originated prior to the origin of the process of pterygoid segmentation; however, it remains ambiguous whether pterygoid segmentation is a synapomorphy of Neornithes, Neognathae, or Neoaves. Overall, our study clarifies the process of avian pterygoid segmentation and raises new questions regarding the major morphological modifications that have characterised the evolutionary history of the avian bony palate.

Purkinje cells (PCs), the sole outputs of the cerebellar cortex, transform granule cell firing patterns into appropriate outputs to drive learning and behavior. Two major PC classes (typically defined by Aldoc expression) can be further subdivided into nine molecularly distinct PC subtypes, suggesting that each subtype might be specialized for distinct categories of cerebellar processing. This has been difficult to test due to limited tools to target PC subtypes. We therefore developed intersectional tools to target PC subtypes, specifically Kcng4+ PCs (primarily Aldoc-) and Gpr176+ PCs (Aldoc1). We mapped their distributions within the cerebellar cortex and quantitatively characterized their outputs onto different types of cerebellar nuclei (CbN) neurons. Although projections by PC subtypes defined by Kcng4 and Gpr176 are regionally segregated within the CbN, each PC subtype synapses onto many types of CbN neurons, and individual CbN neurons often receive convergent inputs from multiple PC subtypes. We also selectively silenced PC subtype outputs and found that silencing Kcng4+ PC outputs impaired motor behaviors while sparing emotional and social behaviors, whereas silencing Gpr176+ PC outputs selectively increased exploratory behavior and reduced anxiety without affecting motor and social behaviors. These findings demonstrate that molecularly defined PC subtypes differentially regulate specific behaviors and establish a versatile framework for uncovering how cerebellar circuits are specialized to control diverse behaviors.

The deepest phylogenetic divergence within crown birds (Neornithes) is that between the reciprocally monophyletic Palaeognathae and Neognathae. Extant palaeognath diversity comprises the iconic flightless "ratites" (ostriches, rhea, kiwi, cassowaries, and emu), as well as 46 species of volant tinamous in Central and South America (Billerman et al., 2020). Although the earliest stages of palaeognath evolution remain shrouded in mystery due to a sparse fossil record, a group of apparently volant extinct palaeognaths from the Paleogene of Europe and North America, the lithornithids, can help to clarify palaeognath origins. Here, we use high resolution microCT scanning to characterize the morphology of two lithornithid specimens from the early Eocene (Ypresian) London Clay Formation: the neotype of Lithornis vulturinus (NHMUK A5204), from the Isle of Sheppey, Kent, England, and a newly discovered clay nodule containing lithornithid postcranial remains from the nearby locality of Seasalter. This three-dimensional dataset reveals bones from the L. vulturinus neotype that are partially or completely covered by matrix, allowing us to redescribe this critical specimen in new detail and present a revised differential diagnosis of L. vulturinus. We refer the new specimen from Seasalter to L. vulturinus on the basis of apomorphies such as a proximally directed lateral process of the coracoid, caudally divergent lateral margins of the sternum, an arcuate deltopectoral crest, as well as its provenance from a nearby penecontemporaneous locality. The Seasalter specimen contains abundant postcranial material that provides new insight into bones damaged or missing in the neotype, including two undamaged scapulae bearing the hooked acromion that is a diagnostic feature of lithornithids, two complete coracoids, and a nearly complete three-dimensionally preserved sternum. Its estimated body mass is one third larger than that of the neotype, indicating intraspecific variation within L. vulturinus that may reflect sexual dimorphism. Molecular divergence dates and Cretaceous neognath fossils indicate the presence of total-clade palaeognaths before the K-Pg mass extinction event; detailed anatomical descriptions of Paleogene palaeognaths will assist in the identification of the first total-clade palaeognaths from the Cretaceous, and provide insight into how and when flight was independently lost among Cenozoic crown palaeognaths.

Life history strategies such as rapid growth and high population turnover rates observed in vertebrates have been thought to have emerged relatively late in evolution. However, very little direct evidence exists at the species level for early vertebrates. In this study, a large fossil collection of over 450 specimens of Protaspis spp, heterostracan agnathans from the Cottonwood Canyon Formation at Beartooth Butte, Wyoming, from the Early Devonian, was analyzed. Morphological observations, analysis of bone plate completeness, and length-frequency analyses using ELEFAN--commonly used in recent fish studies--were applied to reconstruct growth rates, cohort structure, and ontogenetic processes. Protaspis specimens exhibited a continuous growth series from juvenile to adult stages, and a clear cohort structure was identified from the length-frequency distributions. The ELEFAN analysis suggested a life-history characterized by rapid growth and a short life span, and these features remained consistent in subset analyses restricted by species or locality, confirming the robustness of the estimates. Furthermore, the integration of the dermal bone plates progressed during the late stages of ontogeny, revealing that the rapid growth during the juvenile stage preceded the completion of this defensive structure. Comparisons of their growth parameters with those of extant fishes show that Protaspis does not align with slow-growing, long-lived "living fossil" taxa, but instead clusters with small-bodied, fast-growing species. These findings suggest that life-history strategies involving rapid growth and high population turnover were already established in early jawless vertebrates, much earlier than previously assumed.

Learning on both fast and slow timescales is required to enable us to adapt to a dynamic environment. Whether the mechanisms mediating fast and slow learning are implemented by the same, or different, circuit elements remains an important open question. Learning involving the cerebellum is known to be driven primarily by instructive climbing fiber inputs to Purkinje cells, but the dynamics of climbing fibers across learning on fast and slow timescales are not known. It is unclear if climbing fibers encode the same or different instructive signals at different stages of learning, and whether learning-related changes in climbing fiber encoding properties across timescales are driven by the same or distinct mechanisms. We addressed this problem using longitudinal 2-photon calcium imaging of climbing fiber activity across multiple cerebellar lobules as mice learned to execute a visuomotor integration task - slow learning - and then rapidly adapted to a new sensorimotor coupling - fast learning. Instructive signals are spatially segregated in trained mice: Lobule V preferentially encodes movement predictively, while Lobule simplex preferentially receives reward-related feedback. Moreover, the encoding of sensorimotor and reward-related instructive signals is dynamic on both slow and fast timescales: movement-related activity emerges over the course of training, while reward-related activity progressively diminishes as mice become experts but re-emerges specifically in Lobule simplex when rewards become scarce. Finally, while the same climbing fiber inputs can change on both timescales, the magnitude and spatial organization of slow changes are not related to fast changes. Thus, climbing fiber input can carry distinct instructive signals on different timescales - a multiplexing of teaching signals that may underlie the cerebellums capacity to both acquire and refine motor behaviour.

Studies of visual discrimination in rodents can confound the effects of cue salience with reward value, making it difficult to determine which factor guides choice behavior. We examined this issue by testing how changes in relative salience affect decision dynamics in rats performing a two-alternative forced-choice task in which rats chose between visual cues associated with high or low sucrose rewards. After initial training with high and low luminance cues, we introduced a novel cue of intermediate luminance as a "luminance shift" test. The intermediate luminance cue substituted for either the brighter or dimmer cue and had the same reward value as the cue that it replaced. We found that while rats maintained a preference for the higher-value option, the introduction of a perceptually more similar cue consistently reduced choice preference and eliminated latency differences compared to baseline. Using drift diffusion modeling, we determined that the luminance shifts primarily caused a reduction in the drift rate (the speed of evidence accumulation), reflecting increased difficulty in cue discrimination. This finding suggests that the relative salience of the options determines the efficiency of evidence accumulation in value-based decisions. Furthermore, this effect on drift rate shows a dissociation from our previous work (Palmer et al., 2024), where prefrontal cortex inactivation specifically affected only the decision threshold. Our results demonstrate that relative salience influences deliberation, with low-level perceptual features shaping the computational dynamics of value-based choice. Our findings clarify the distinct contributions of sensory input and prefrontal function in the decision process. Significance StatementThis study reveals that changes in the relative salience of visual stimuli shape the computational dynamics of value-based decisions. We trained rats to make visually guided choices and found that relative differences in the brightness of the stimuli affect how quickly the rats made decisions and how often they chose a higher-value option. Our findings, together with a recent study on the role of the prefrontal cortex in value-guided decisions (Palmer et al., 2024), suggest that separate factors influence choice dynamics in rodents: visual salience affects the speed of deliberation, while prefrontal activity regulates caution. This study helps clarify how sensory and higher cognitive variables relate to the distinct computational components of the decision process.

Motor adaptation involves the parallel operation of implicit recalibration and explicit re-aiming processes. During naturalistic learning, these systems interact, producing compound behavioral outputs that reflect their combined contributions. It remains unclear whether simultaneously engaged implicit and explicit processes form a single unified representation, or generate parallel memory representations that are merely co-expressed, and how consolidation transforms such representations. We addressed these questions across three visuomotor adaptation experiments (n = 120), in which the implicit process was engaged via gradual cursor rotation and the explicit process via target jump, by systematically manipulating the sequence of learning and the timing of expression. Immediately after learning, behavior reflected an inflexible, integrated memory that could not be decomposed by changing task demands. Following 24-hour consolidation, however, expression became component-selective, with implicit or explicit contributions retrieved in response to task demand. This reorganization had direct consequences on relearning, producing facilitation when the expressed and relearned components matched and interference when they mismatched. Moreover, when implicit adaptation was stabilized prior to compound learning, consolidation preserved the updated state rather than the original implicit representation. Together, these findings demonstrate that consolidation does not merely stabilize compound motor memories. Instead, it actively reorganizes them, transforming the initially integrated representations into independent, context-dependent components.

T cells can respond to even a single molecular binding event of antigen to a TCR. A key step in the TCR signaling pathway that definitively exhibits this single molecule response is the initiation of calcium influx by activation of PLC{gamma}1 in the LAT protein condensate. Here, we describe detailed kinetic measurements examining how protein condensation of LAT regulates activation of PLC{gamma}1 using a reconstituted membrane system. The results reveal that membrane recruitment of PLC{gamma}1 is tightly controlled by the LAT phosphorylation state, with no measurable independent recruitment to PIP2 or PIP3 lipids via the PLC{gamma}1 PH domains. We further observe PLC{gamma}1 is rapidly activated by membrane-associated kinase upon recruitment, irrespective of the LAT condensation state. These studies also revealed a crosstalk mechanism in which the TEC family kinases responsible for PLC{gamma}1 activation also phosphorylate LAT. This interaction establishes a positive feedback loop in LAT phosphorylation, mediated through LAT condensation, which drives a bimodal LAT phosphorylation response to TCR activation. Kinetic modeling reveals how this LAT phosphorylation response can cooperatively gate PLC{gamma}1 activation from a single TCR. These results suggest the LAT condensate facilitates both signal amplification and noise suppression in PLC{gamma}1 activation through a bimodal switch affecting LAT phosphorylation. Significance StatementT cells are sensitive sensors capable of detecting and responding to trace amounts of foreign antigen. Understanding how they achieve such sensitivity while maintaining accurate antigen discrimination remains a key challenge. Here, through detailed kinetic measurements of PLC{gamma}1 activation, we identify a cross reactivity in which kinases responsible for PLC{gamma}1 phosphorylation also phosphorylate LAT. This creates a bimodal switch controlling LAT phosphorylation levels, which gates PLC{gamma}1 activation from single TCR signals. We suggest this mechanism plays a key role in the signal amplification and noise suppression required for T cells to detect single antigen molecules.

Interference between consecutively acquired motor memories is a defining feature of sensorimotor adaptation, yet its mechanistic origin remains unresolved. Because adaptation is supported by separable explicit strategies and implicit recalibration, it offers a means to identify the learning process that gives rise to interference. Across four visuomotor adaptation experiments, we examined the conditions under which the acquisition of a new, competing motor memory influences the expression of a previously acquired memory. We selectively biased new learning towards either explicit or implicit processes, and quantified its impact on the recall of the original memory 24-hours later. Under standard adaptation conditions, participants exhibited classic interference, such that re-learning was indistinguishable from naive performance. However, when new learning was driven primarily by explicit strategies induced through delayed endpoint feedback, interference was markedly attenuated and the original memory was preserved. In contrast, when the competing memory was implicitly forged under error-clamp conditions, robust interference emerged. Furthermore, disrupting posterior parietal cortex (PPC) with cathodal hd-tDCS prior to implicit learning attenuated interference, indicating that intact PPC processing is required for incorporating new learning into an existing sensorimotor representation. Taken together, these findings suggest that interference reflects the integration of new learning into a shared representational substrate via implicit recalibration, a process that limits the coexistence of competing motor memories.

The subiculum (SUB) is the main output structure of the hippocampus, influencing diverse behaviors through its widespread cortical and subcortical connections. Our previous work creating the mouse Hippocampus Gene Expression Atlas (HGEA) identified four genetically distinct cellular layers across five columnar domains in the SUB, with gene expression boundaries corresponding to distinct connectivity patterns and brain-wide networks involved in spatial navigation, social behavior, and neuroendocrine regulation (Bienkowski et al., 2018). Using the Digital Brain Mouse Projectome Atlas (MPA) tool, we conducted virtual tract-tracing to assess whether connectivity patterns of single-neuron 3D reconstructions aligned with HGEA-defined SUB cell types (Qiu et al., 2024). We classified 689 SUB projection neurons into 12 HGEA cell-type groups based on their laminar and columnar distributions, whose spatial organization recapitulated HGEA-defined 3D boundaries. Using this population sample, we performed a SUB cell-type census, characterized neuronal heterogeneity and projection prevalence, identified common and rare connectivity motifs and axonal collateralization patterns, and defined distinct projection themes for each SUB cell type. Together, this analysis integrates single-neuron and population-level data to advance understanding of SUB cell type organization and its contributions to brain-wide networks regulating diverse behaviors.

Smell allows animals to find food, avoid danger, and communicate through the binding of odorants to chemosensory receptors on olfactory sensory neurons. The vision-priority hypothesis predicts an antagonistic relationship between olfaction and vision, in which olfactory ability increases as visual acuity decreases along evolutionary lineages, a tradeoff that often occurs through expansion and contraction of chemosensory receptor gene families. The Mexican tetra (Astyanax mexicanus), a fish species with both sighted surface-dwelling and blind cave-dwelling populations, presents an ideal model for exploring the mechanisms underlying this tradeoff. Here we show that although cavefish can sense odorants at lower concentrations than surface fish, they do not have an expanded repertoire of chemosensory receptors, increased sensory neuron number or density, or enhanced expression of receptors compared to surface fish. Instead, cavefish have physiological adaptations to the olfactory epithelium, including more motile cilia and decreased flow rate through the olfactory pits. Pharmacological attenuation of flow rate in the olfactory pits in surface fish increased visits to the odorant source, suggesting that the reduced flow rate in cavefish is an adaptation leading to better foraging. This unexpected evolutionary path to enhanced olfaction as a compensation for loss of vision underscores the need for mechanistic understanding of comparative genomics.

The rostral nucleus of the solitary tract (rNST) is the initial central site for taste processing. This nucleus has a complex circuitry and multiple cell types with different response properties, connectivity, and morphology (Travers and Travers 2018). However, unlike its visceral counterpart, the caudal NST, neurochemical phenotypes in rNST are poorly defined. Recent studies have begun to probe this gap. Based on fiber photometry, optogenetics, and cell-type specific deletion. For example, one group proposed that somatostatin (SST) rNST neurons, neither calbindin or dynorphin cells, responded specifically to bitter stimuli and that these neurons were necessary for suppression of quinine-induced licking (Jin, Fishman et al. 2021) (Zhang, Jin et al. 2019). The present study employed in situ hybridization, optotagging, and chemogenetic suppression in male and female mice to demonstrate that SST neuron function is more complex. Although most SST neurons responded optimally to bitter stimuli, many others were activated by different qualities and some non-SST neurons responded to bitter tastants. Moreover, roughly equal proportions of SST neurons expressed excitatory (VGLUT2) or inhibitory (VGAT) markers. Suppressing SST neural activity with DREADDS enhanced licking to both quinine and sucrose suggesting that neural activity elicited by the aversive bitter stimulus was suppressed whereas licking elicited by the sweet, preferred stimulus was increased. We hypothesize that these effects arise from suppressing excitatory quinine-responsive SST neurons but that a separate population of inhibitory SST neurons synapse on sucrose-responsive cells. Significance StatementRecent studies have revealed molecular heterogeneity of gustatory system neurons. However, it is unclear whether molecularly-distinct cells are associated with specific roles. The current study investigated somatostatin (SST) neurons in rNST, the first central hub for taste processing. Well over half were inhibitory, expressing VGAT, but a substantial proportion were excitatory, expressing VGLUT2. A narrow majority responded optimally to the bitter quality and none to NaCl, but other SST cells responded most vigorously to sweet, umami, or sour stimuli. Subsets of neurons not expressing SST responded best to each quality, including bitter. Suppressing activity in SST neurons dampened behavioral avoidance to quinine but enhanced consummatory responses to sucrose. Thus, SST rNST neurons exhibited varied functional characteristics but also clear distinctiveness.

The neuropeptide signaling pathway is vital for the physiology and behavior of multicellular organisms. This pathway is mediated by ligand-receptor binding, wherein neuropeptides (NPs) are often released from neurons, while their receptors (NPRs) are ubiquitously expressed in both neuronal and non-neuronal cell types. To elucidate the underlying mechanisms of this expression pattern divergence, here we dissect the genomic and epigenomic architectures of these two distinct gene classes. We first characterized the genomic architecture of NP and NPR genes to compare their total gene length, exon and intron lengths, exon counts, and number of alternatively spliced transcripts per gene. We also profiled their regulatory genomic elements including CpG islands, TATA boxes, and their overlapping antisense transcripts. These analyses were then expanded to non-human model organisms to evaluate the evolutionary conservation of the underlying mechanisms. We found that NPRs encompass larger genomic loci and encode longer transcripts than NPs. Furthermore, the increased length of NPR transcripts was driven by longer exons rather than higher exon counts. Consistently, the number of spliced variants per gene was similar between NPs and NPRs, suggesting that alternative splicing events are a minor contributor to their distinct expression patterns. At the RNA level, NPR mRNAs possess significantly longer 3'UTRs compared to NPs, indicating a greater potential for post-transcriptional gene regulation. This complexity is also mirrored at the chromatin level, where NPR loci exhibit a higher density of epigenetic marks than NPs. Together, these findings highlight the multi-layered nature of gene regulation prioritizing control at the receptor level.