Activity-dependent structural plasticity is essential for the growth and remodeling of synaptic connections. At the Drosophila melanogaster larval neuromuscular junction (NMJ), spaced stimulation induces the translation-dependent formation of ghost boutons (GBs), which are immature boutons that lack a corresponding postsynaptic structure. Calcium/calmodulin-dependent protein kinase II (CaMKII) has previously been implicated in GB formation and is locally translated at synapses, raising the possibility that activity-dependent CaMKII synthesis contributes to GB formation. We examined activity-induced synaptic outgrowth in female larvae using a spaced depolarization paradigm combined with genetic manipulations of the endogenous CaMKII locus. While whole-animal CaMKII null mutants exhibited fewer GBs after spaced depolarization, selective disruption of activity-dependent CaMKII synthesis by deletion of the CaMKII 3′ untranslated region (3′UTR) in either presynaptic motor neurons or postsynaptic muscle cells had no effect on GB formation. Consistent with this, inhibition of the signaling pathways upstream of CaMKII synthesis also did not impair bouton outgrowth. Cell-specific deletion of the CaMKII coding region in presynaptic neurons also did not alter GB formation, but postsynaptic deletion significantly reduced it. These findings demonstrate that local synthesis of CaMKII is dispensable for activity-dependent GB formation. Instead, our results indicate that only steady-state CaMKII protein is necessary for this specific type of structural plasticity and identifies a new transsynaptic function for postsynaptic CaMKII in GB formation. These findings distinguish the role of CaMKII protein from CaMKII synthesis and suggest that other locally translated proteins underlie the protein synthesis dependence of GB formation.

Humans exhibit machine-like eye movements (consistent and repeatable) in space and time while performing demanding acuity tasks. To investigate these, we used an adaptive optics imaging and display system in 6 human subjects (4 females, 2 males) to present ultra-sharp Vernier acuity stimuli briefly every 2 s while simultaneously measuring eye movements, including precisely where on the retina each stimulus fell. We found that drifts and microsaccades combined to confine the landing location of the anticipated stimulus to a tiny retinal region centered on the preferred retinal locus (PRL). The variance of landing location was smallest at the time of stimulus presentation and a few hundred milliseconds after. We correlated where the stimulus fell in space and time with correct or incorrect responses. The PRL and a small area around it, including the anatomical fovea, conferred the best acuity. Acuity declined consistently in the rare events in which the stimulus fell more than 5 minarc from the PRL. We also found that acuity was best when the last microsaccade occurred sufficiently prior to stimulus presentation. Our findings reveal a highly evolved oculomotor system where gaze direction during fixation is rarely far enough from the PRL to compromise visual resolution when a person makes natural fixational eye movements.

The lateral reticular nucleus (LRN) is positioned to relay motor-related information to cerebellar circuits, but its direct contribution to skilled reaching and grasping remains unclear. Here, we examined skilled forelimb behavior in intact adult female Long–Evans rats after bilateral, cell type-targeted LRN ablation using single-pellet reaching, qualitative scoring, and quantitative three-dimensional kinematic analysis. We assessed whether LRN loss disrupted gross limb transport, endpoint precision, trial-to-trial consistency, and reach timing across pre- and post-surgical recording sessions. LRN-ablated animals continued to generate broadly recognizable pellet-directed reaches, indicating that the LRN is not required for basic reach production. However, group differences emerged in restricted portions of the reach trajectory and were most prominent in pellet-directed endpoint control. Experimental animals showed broader endpoint covariance, greater endpoint spread, and increased trial-to-trial variability, indicating less precise and less reproducible forelimb placement relative to the pellet. These effects were not explained by a single fixed spatial offset, but instead reflected reduced endpoint stabilization accompanied by selective coordinate-specific changes, including altered paw height. Reach duration was also altered, but these timing differences emerged later and were less prominent than the spatial endpoint deficits. Together, these findings suggest that the LRN contributes primarily to the refinement, stabilization, and timing of skilled forelimb movements rather than to gross reach initiation or limb transport. This work provides a direct in vivo model for studying LRN-dependent control of skilled reaching and highlights the value of kinematic analysis for detecting subtle movement deficits beyond retrieval success alone.

Each barrel of the barrel cortex (BC) receives thalamic input primarily from a single whisker. Barrels then communicate through intracortical circuitry to integrate input from multiple whiskers and perform processing functions such as spatial filtering, motion sensing, and object localization. To investigate the circuits that enable barrels to communicate independently of extracortical and inter-areal influences, we prepared slices of somatosensory cortex from mice of both sexes with a hybrid voltage sensor (hVOS) targeted to Scnn1a excitatory neurons in cortical layer 4 (L4). We then imaged voltage responses of this population of neurons to electrical stimulation. Imaging tracked activity initiated in L4 of one barrel spreading into layer 2/3 (L2/3) and then to L4 of neighboring barrels. AMPA receptor blockade eliminated this spread, suggesting that L4 signaling to neighboring barrels employs an L4→L2/3→L4 excitatory synaptic relay. Blocking AMPA receptors also enhanced some responses, revealing intra- and inter-barrel feedforward inhibition. Both coronal and sagittal slices presented the layout of barrels, which aligned with facial whisker organization. We assessed inter-barrel communication in different directions and found it to be isotropic in response amplitude, half-width, and conduction velocity. However, latency was longest for communication to caudal barrels. Furthermore, paired-pulse depression was strongest and recovery slowest for inter-barrel communication related to exploratory protraction. Such biases have the potential to contribute to direction-sensitive processing. Anisotropy in short-term plasticity can tune BC microcircuits to preserve temporal fidelity and filter selectively according to direction.

Ambient luminance varies widely, and retinal output is shaped by adaptation-dependent gain control. Relative to the adaptation level, a decrease in light intensity can either return intensity toward the adaptation level or drive it farther away from that level. Whether OFF retinal outputs signal both changes simply as light-intensity decrements or distinguish them according to their relationship to the adaptation level is currently unclear. This study examined OFF sustained retinal ganglion cells in bullfrogs ( Rana catesbeiana ) of either sex and quantified response bias by comparing spike counts during decrements that moved toward and away from the adaptation level. Responses fell into two functional populations: one signaled decrements in both cases, whereas the other selectively signaled decrements that moved away from the adaptation level, indicating adaptation level-referenced encoding. Although surround suppression was considered a possible contributor to these differences, manipulations expected to reduce classical surround contributions did not explain the selective suppression of responses during decrements that moved toward the adaptation level. Blocking GABAA receptors (GABAARs) consistently weakened the response asymmetry between decrements that moved toward and away from the adaptation level, suggesting that GABAAR-dependent inhibition contributes to the mechanism underlying adaptation level-referenced responses. Taken together, these findings reveal a distinct computation for light-intensity decrement processing and expand the functional diversity of OFF outputs across light-intensity contexts.

Despite a wealth of information provided by face perception research, the neural processes by which unfamiliar faces become familiar—particularly via passive exposure—remain poorly understood. Here, we introduce a novel fast periodic visual stimulation electroencephalography (FPVS-EEG) paradigm designed to objectively track neural familiarization. Specifically, our paradigm measures the emergence of implicit face-identity familiarity during brief, passive repetition learning, without requiring explicit recognition or task engagement. The paradigm uses ecologically valid stimuli incorporating image variations characteristic of forensic material. We tested 28 (18 females) healthy human volunteers including neurotypical civilians and law enforcement professionals, all with typical face-processing abilities. Neural responses were measured to a 1 Hz tagged oddball identity embedded within a 6 Hz stream of changing base images, using high-quality mugshot and CCTV-like stimuli. Results showed that neural signatures of implicit familiarity can emerge rapidly within minutes of passive exposure but only when high-quality mugshot images are used. These familiarity-related responses were characterized by concurrent neural suppression and enhancement, likely to reflect the co-occurrence of repetition detection and identity learning processes. The FPVS-EEG approach reported here provides an objective and quantifiable measure of implicit, early face learning that could complement behavioral assessments and inform future research on individual differences and applied contexts.

Attention is a powerful mechanism that dynamically optimizes brain representations to prioritize behaviorally relevant information. While previous studies focusing on single tasks have demonstrated that attention shifts tuning toward attended targets, real-world behavior often requires humans to switch between tasks with different demands. How does visual-semantic tuning in the human brain shift to support the behavioral demands of different naturalistic tasks? To investigate this question, we used voxelwise encoding models to explore how visual-semantic tuning across the human cerebral cortex shifts between two naturalistic human fMRI experiments that used distinct tasks, movie-watching (five male participants) and active navigation (six participants, three male, three female). Results show that visual-semantic tuning in the cortex differed substantially between experiments. Principal component analysis reveals that during navigation, tuning shifts increase the representation of vehicles and traffic signs compared with movie-watching and that these shifts are localized to distinct functional networks. These tuning shifts reconfigure the human brain’s representations of object categories based on their behavioral relevance. These findings suggest that visual-semantic tuning in the brain dynamically shifts toward task-relevant information across naturalistic tasks, optimizing functional representations to achieve diverse behavioral goals.

The past few years have seen an increase in the use of encoding models to explain neural responses to natural speech. The goal of these models is to characterize how the human brain converts acoustic energy into distinct linguistic representations that enable everyday speech comprehension. For example, researchers have shown that electroencephalography (EEG) data can be modeled in terms of acoustic features of speech, such as its amplitude envelope or spectrogram, linguistic features such as phonemes and phoneme probability, and higher-level linguistic features like context-based word predictability. However, it is unclear how reliably EEG indices of these speech feature representations reflect comprehension in different listening conditions. To address this, we recorded EEG from 25 neurotypical adults (nine males) who listened to segments of an audiobook in various levels of background noise. We modeled how their EEG responses reflected a range of acoustic and linguistic speech features and how this tracking varied with behavior across noise levels. EEG tracking of nearly all examined features showed SNR-dependent changes in unique variance explained, with the largest changes occurring for linguistic features. We hypothesized that only higher-level feature tracking would predict behavior but instead found that both high- and low-level features were associated with behavioral scores depending on the noise level. EEG markers of the influence of top–down, context-based prediction on bottom–up acoustic processing also correlated with behavior. These findings help characterize the relationship between brain and behavior by comprehensively linking hierarchical indices of neural speech processing to language comprehension metrics.

The auditory brainstem plays a crucial role in speech-in-noise (SiN) listening, refining numerous acoustic features such as pitch and spatial location under continual descending influence from the cortex. However, the difficulty of characterizing brainstem activity during continuous speech listening has obscured its functional role in ecologically valid contexts—not only the effects of selective attention on neural responses but also their impact on comprehension and listening effort. Here, we evaluated the role of the brainstem in SiN perception and other listening behaviors using a continuous, speech-based stimulus with embedded chirps (Cheech) that rapidly and effectively evoked auditory brainstem responses (ABRs) while 25 participants listened to short story narratives ( n  = 15 females, 10 males). The Cheech-modified stories were presented alone or in the presence of another spatially separated talker, and neural responses were measured throughout by EEG. Both word-level detection performance and narrative-level comprehension were evaluated, as well as subjective reports of listening effort. ABR wave V was modulated by the presence of a competing talker. Additionally, wave V peak amplitudes were associated with identification speed of the target words embedded within the stories, while faster latencies were related to better target word identification accuracy, comprehension question accuracy, and lower subjective listening effort. Collectively, our results provide evidence for the influence of brainstem encoding processes on individual speech-listening behaviors, including the ability to selectively attend to and comprehend target speech in the presence of a competing talker.

Animals must flexibly discriminate between threat and nonthreat to deploy adaptive defensive strategies. We introduce a single-session differential signaled active avoidance paradigm that temporally dissociates acquisition, consolidation, and retrieval of instrumental avoidance memory. Interleaving a behaviorally noncontingent neutral cue (CS−) with a threat-predictive, behaviorally contingent cue (CS+) enhanced long-term memory without altering acquisition, demonstrating that differential contingency structure selectively reinforces memory consolidation rather than influencing learning performance. Naive mice of either sex inferred contingencies within a single session, and discrimination achieved during training predicted retrieval precision. However, discrimination operated within defined boundary conditions: Extended training or elevated threat intensity destabilized cue specificity and promoted persistent avoidance generalization. Under high-threat conditions, freezing and shuttling coemerged as complementary defensive responses, indicating a shift from precise cue-based encoding to a generalized defensive state. Remote retrieval recruited oxytocin receptor-expressing cells in the medial prefrontal cortex and activated mTORC1-dependent translational signaling, implicating protein synthesis in maintenance of discriminative avoidance memory. In a tuberous sclerosis complex mouse model with Tsc2 haploinsufficiency in oxytocin-responsive cells, males displayed intact acquisition but generalized avoidance at both recent and remote time points, a deficit not rescued by additional training. These findings identify oxytocin-modulated translational control as a molecular gate stabilizing threat-safety discrimination and show that disruption of this axis—by excessive threat or reduced Tsc2 gene dosage—biases memory toward pathological generalization, providing a mechanistic framework for safety-learning deficits in neurodevelopmental and anxiety-related disorders.

Humans can experience sound-evoked pain, either from extremely loud sounds or in cases of pain hyperacusis from typically tolerable sounds. However, the mechanisms underlying sound-evoked pain remain poorly understood. Developing behavioral methods to measure sound-evoked pain in animal models is critical for elucidating these mechanisms. Here, a machine learning-based approach was developed to measure sound-evoked pain in freely moving mice of both sexes by analyzing facial grimace and body position from video recordings during sound exposure. Facial grimace, a commonly used method to detect pain in mice, and body position, which can be used to measure postural and movement changes that also indicate pain, were both quantified using a deep neural network model trained to extract established facial and body features from video recorded by a single camera. To validate the model's capability to detect pain, a known painful state, migraine induced by the injection of the neuropeptide calcitonin gene-related peptide (CGRP), was used. Using this machine learning-based approach, the ability to quantify a pain response from CGRP-induced migraine, distinct from baseline behavior, was demonstrated, resulting in a defined pain threshold. Sound exposures at high intensities elicited significant changes in facial grimace and body position, in comparison, surpassing the pain threshold calculated from CGRP-induced migraine. These behavioral changes were absent in Tmie -knock-out mice, which lack functional sound transduction in the cochlea. This automated, high-throughput framework enables objective and sensitive analysis of pain-related behavior providing a foundation for future studies investigating the peripheral and central mechanisms of sound-evoked pain.

Chronic stress exposure causes neurobiological and behavioral changes that resemble those reported in psychiatric conditions such as major depressive disorder (MDD). Preclinical stress models and studies using postmortem tissue from MDD patients have shown that DNA damage-inducible transcript 4 ( Ddit4 ) is increased in the prefrontal cortex (PFC). This is important because DDIT4 negatively regulates the mammalian target of rapamycin (mTOR) pathway, which may lead to behavioral deficits through diminished neuroplasticity and PFC function. Our prior studies indicate that coordinated neuron–microglia interactions contribute to synaptic remodeling in the PFC. The present studies aimed to test the hypothesis that increased neuronal Ddit4 expression is sufficient to drive structural remodeling of PFC neurons, in part by provoking microglia activation, and this leads to behavioral and cognitive deficits. To this end, we bilaterally infused AAV5-hSyn1-Ddit4-tdTomato or a control vector into the PFC of male Thy1-GFP and C57BL/6 mice and examined molecular, cellular, and behavioral endpoints. Mice with Ddit4 overexpression (Ddit4-OV) showed no change in passive stress coping, yet exhibited deficits in temporal order memory. Immunohistology analyses showed a decrease in dendritic spine density of Ddit4-OV mice. However, we found no changes in microglia count, microglia size, or nearest neighbor distance. Bulk RNA sequencing of Ddit4-OV PFC revealed increases in transcripts involved with dendrite and synapse function and decreases in transcripts involved with mitochondrial function, implicating mTOR dysregulation. Altogether, these results indicate that Ddit4 overexpression recapitulates some of the broad molecular, cellular, and behavioral adaptations observed following chronic stress exposure through a cell-autonomous mechanism.

The paraventricular nucleus of the thalamus (PVT) has emerged as an important node in circuits regulating motivated behavior. The neuronal pathway from the lateral hypothalamus (LH) to the PVT has been shown to regulate arousal, feeding, and reward seeking. However, the involvement of the LH–PVT pathway in individual differences in cue-motivated behavior remains unclear. During a pavlovian conditioned approach (PavCA) paradigm, when a reward is repeatedly preceded by the presentation of a cue, rats come to exhibit a conditioned response to the cue. One extreme of the population, sign-trackers (STs), approaches and interacts with the cue itself, while the other extreme, goal-trackers (GTs), approaches the location of reward delivery. Intermediate responders (IRs) approach and interact with both the cue and reward location, without a preference. We utilized a PavCA paradigm in male rats to examine the effects of LH–PVT pathway inhibition on individual differences in cue-motivated behavior. A dual-vector approach was used to express inhibitory chemogenetic receptors in the LH–PVT pathway. We found that inhibition of the LH–PVT pathway selectively attenuates the expression of goal-tracking behavior, without affecting sign-tracking. This effect is driven primarily by IR rats, as inhibition of LH–PVT neurons attenuates goal-tracking behavior in IRs, without impacting the response of STs or GTs. We speculate that the flexibility of responding in IR rats made them especially vulnerable to this manipulation. These findings identify the LH–PVT pathway as a selective contributor to reward-directed conditioned responding and a circuit substrate for behavioral flexibility.

Humans can flexibly acquire entirely new sensorimotor mappings, a process known as de novo motor learning. A central challenge in de novo motor learning is that the learner must discover a viable solution from scratch within a highly redundant control space, without predefined task constraints. Understanding what types of sensorimotor information contribute to the formation of accurate motor behavior in such situations is therefore critical for explaining how novel sensorimotor skills are acquired. While previous studies have suggested that novel visuomotor mappings can be formed based on movement direction and target position, it remains unclear how these two types of information contribute to the learning process. To address this question, we trained 25 human participants (13 males and 12 females) to learn arbitrary joystick-to-cursor mapping. We then employed a generalization paradigm to selectively restrict learning experience to either movement direction or target position. Three distinct target conditions were designed: one emphasized target position (P), another emphasized movement direction (D), and a third (P&D) encouraged learning of both components separately. Results showed direction experience reduced initial direction error, whereas the lack of position experience was associated with increased final position error. However, in the P&D condition, combining these experiences did not yield additive generalization. Instead, endpoint accuracy was positively correlated with the alignment between direction- and position-based outputs in the redundant control space. These results suggest that accurate formation of a novel sensorimotor map depends on the coordinated use of directional and positional experiences.

Visual scene analysis relies on a set of scene-selective regions in the posterior cerebral cortex (OPA, PPA, MPA), with a paired memory-responsive counterpart located in the anterior (LPMA, VPMA) or overlapping (MPMA) cortex. The interaction between these pairs of regions is thought to integrate visual input with mnemonic context. Recently, a fourth scene perception area in the superior parietal cortex (the superior place area, SPA) was identified, with a proposed role in visually guided navigation. Whether this region also has an anterior paired memory region is currently unknown. Across two independent fMRI datasets (total N  = 24, 14 females) using static or dynamic stimuli and distinct memory tasks, we show that recalling visual scenes evokes robust responses in a region immediately anterior and dorsal to SPA (referred to here as the superior place memory area, SPMA). During resting-state fMRI, SPA preferentially coupled with the other scene perception areas, while SPMA preferentially coupled with the other place memory areas. At the whole-brain level, seed-based connectivity revealed that SPA sits at the confluence of four processing streams spanning regions implicated in egocentric scene perception, map-based navigation, perspective taking, and goal-directed movement. These findings extend the perception–memory motif associated with visual scene processing to a fourth cortical surface. The widespread anatomical coupling between scene perception and memory processes reflects the importance of this interaction for flexible, context-grounded navigation.

Stress is a major risk factor for various psychiatric disorders. Activation of the hypothalamic–pituitary–adrenal axis increases cortisol release, whereas prolonged glucocorticoid exposure has been linked to cognitive impairment and mood disorders. In this study, we examined whether chronic corticosterone administration alters emotional states, active coping strategies, and associated neuronal activation in rats. Male Long–Evans rats were trained in a lever-press task to avoid signaled footshocks and then assigned into control or corticosterone-treated groups, with the latter receiving daily corticosterone injections for 21 consecutive days. Afterward, emotional states were assessed using the elevated plus maze (EPM), forced swim test (FST), and sucrose preference test (SPT). Finally, the animals were retested for their active avoidance behavior, while neuronal activation levels in related brain areas were evaluated. Our results showed that chronic corticosterone administration led to decrease in body weight and deteriorated fur condition over time. The stressed rats showed hypolocomotion in EPM but no changes in anxiety-like behavior, increased despair-like behavior in FST, and developed anhedonia in SPT. Despite these emotional alterations, active avoidance performance and freezing behavior were not affected. Consistently, analyses of numbers of c-Fos–positive neurons in the prelimbic cortex, infralimbic cortex, nucleus accumbens core, nucleus accumbens shell, dorsal CA3, and basolateral amygdala did not differ between groups. Together, our results suggested that chronic corticosterone administration induced physical deterioration. These animals were less active and developed behavioral despair and anhedonia, but did not alter their coping strategies to threat-related signals.

Distinct neocortical regions subserve different sensory functions, yet the cellular features that distinguish neurons across cortical areas remain poorly understood. Layer (L)2/3 pyramidal neurons (PyNs) are generated at similar developmental times throughout the cortex, but whether their morphological and functional properties are shaped by areal identity programs is unclear. Here, we compared L2/3 PyNs in mouse primary somatosensory (S1) and primary visual (V1) cortices (both male and female). We observed pronounced areal differences where V1 neurons exhibited smaller and less complex dendritic arbors and displayed increased intrinsic excitability relative to their counterparts in S1. These differences were present in both juvenile and adult stages, indicating that they emerge early and persist over time. Given prior evidence implicating the transcription factor LHX2 in dendritic arborization, we tested its contribution to these differences. Loss of Lhx2 in E15.5 V1 progenitors did not alter neuronal morphology in V1, in contrast to our previous findings in S1. We found that E15.5 progenitors display lower levels of LHX2 protein in V1 than in S1; therefore, we overexpressed Lhx2 in V1 progenitors and found increased dendritic branching complexity in V1 L2/3 PyNs. Together, these findings identify LHX2 as a molecular regulator that contributes to area-specific structural differentiation of L2/3 PyNs. Our results show that PyNs arising from different regions of the dorsal pallium diverge in morphology and physiology according to the cortical area, suggesting that regionally patterned transcriptional programs help establish functional specialization across the neocortex.

Persistent avoidance plays a major role in maintaining anxiety disorders. The few studies that have explored the neural basis of avoidance have predominantly examined established avoidance behaviors rather than the learning processes that establish avoidance responses. We aimed to characterize the temporal dynamics and neural mechanisms of avoidance learning using a novel paradigm combined with 7 tesla fMRI in 82 healthy participants (37 male and 45 female). Participants were first conditioned to two threat stimuli and a compound safety stimulus. Next, they were trained to avoid one threat stimulus while the other remained unavoidable. Behavioral data confirmed accurate learning of task contingencies. Successful avoidance was associated with prominent activation of the ventromedial prefrontal cortex (vmPFC), posterior cingulate cortex (PCC), and ventrolateral prefrontal cortex compared with unsuccessful avoidance, with additional nucleus accumbens and orbitofrontal cortex engagement when compared with safety learning ( P FDR whole-brain corrected). Critically, early compared with late avoidance learning was distinctly associated with activation of midbrain-striatal circuits, including the periaqueductal gray (PAG), mediodorsal thalamus, substantia nigra, and anterior insular cortex—regions associated with threat processing and learning-related signaling. These findings suggest that initial avoidance learning relies on subcortical threat-responsive and learning-related circuits, whereas established avoidance responses engage cortical safety-processing regions. This dissociation parallels recent findings in safety learning and provides novel mechanistic insights for interventions targeting maladaptive avoidance patterns.

The cerebellum, a major structure of the vertebrate central nervous system, forms the dorsal region of the hindbrain. While evidence from connectomics, neuron classification, and gene expression analyses suggests that the major CNS divisions of forebrain, midbrain, hindbrain, and spinal cord predate the vertebrate/tunicate divergence, it is unknown if the subdivision of the hindbrain into a cerebellum, or cerebellum precursor structure, does as well. In fact, conflicting views exist over whether even the most basal vertebrates, the cyclostomes, have a cerebellum homolog. We report here that the motor ganglion (MG), a structure that has been homologized to the vertebrate hindbrain in the chordate Ciona (a tunicate), has functionally distinct dorsal and ventral domains, similar to the generalized hindbrain of vertebrates. In Ciona, the dorsal MG functions as an ascending relay and processing center for peripheral sensory neurons, while the ventral domain processes and relays descending sensorimotor commands. These differences are reflected in distinct neuron classes present in the two domains, as well as in gene expression. While the dorsal MG of Ciona and HB of vertebrates differ in function and complexity, they share a number of similarities. Thus, while it is likely that the cerebellum proper evolved within vertebrates, our findings support the hypothesis that a simpler, dorsally positioned antecedent was present before the tunicate–vertebrate divergence.