bioRxiv Subject Collection: Neuroscience's Journal

"Micro-offline gains" convey no benefit for motor skill learning
While practising a new motor skill, resting for a few seconds can improve performance immediately after the rest. This improvement has been interpreted as rapid offline learning1,2 ('micro-offline gains', MOG), supported by neural replay of the trained movement sequence during rest3. Here, we provide evidence that MOG reflect transient performance benefits, partially mediated by motor planning, and not replay-mediated offline learning. In five experiments, participants trained to produce a sequence of finger movements as many times as possible during fixed-duration practice periods. When participants trained during 10-second practice periods, each followed by a 10-second rest period, they produced more correct keypresses during training than participants who trained without taking breaks. However, this benefit vanished within seconds after the end of training, when both groups performed under comparable conditions, revealing similar levels of skill acquisition. This challenges the idea that MOG reflect offline learning, which, if present, should result in sustained performance benefits, compared to training without breaks. Furthermore, sequence-specific replay was not necessary for MOG, given that we observed persistent MOG when participants produced sequences that never repeated, preventing any effect of (sequence-specific) replay on the performance of subsequent (novel) sequences. Importantly, we observed diminished MOG when participants could not pre-plan the first few movements of an upcoming practice period. We conclude that 'micro-offline gains' represent short-lived performance benefits that are partially driven by motor pre-planning, rather than replay-mediated offline learning.

On the validity of electric brain signal predictions based on population firing rates
Neural activity at the population level is commonly studied experimentally through measurements of electric brain signals like local field potentials (LFPs), or electroencephalography (EEG) signals. To allow for comparison between observed and simulated neural activity it is therefore important that simulations of neural activity can accurately predict these brain signals. Simulations of neural activity at the population level often rely on point-neuron network models or firing-rate models. While these simplified representations of neural activity are computationally efficient, they lack the explicit spatial information needed for calculating LFP/EEG signals. Different heuristic approaches have been suggested for overcoming this limitation, but the accuracy of these approaches has not fully been assessed. One such heuristic approach, the so-called kernel method, has previously been applied with promising results and has the additional advantage of being well-grounded in the biophysics underlying electric brain signal generation. It is based on calculating rate-to-LFP/EEG kernels for each synaptic pathway in a network model, after which LFP/EEG signals can be obtained directly from population firing rates. This amounts to a massive reduction in the computational effort of calculating brain signals because the brain signals are calculated for each population instead of for each neuron. Here, we investigate how and when the kernel method can be expected to work, and present a theoretical framework for predicting its accuracy. We show that the relative error of the brain signal predictions is a function of the single-cell kernel heterogeneity and the spike-train correlations. Finally, we demonstrate that the kernel method is most accurate for the dominating brain signal contributions. We thereby further establish the kernel method as a promising approach for calculating electric brain signals from large-scale neural simulations.

Ultraslow serotonin oscillations in the hippocampus delineate substates across NREM and waking
Beyond the vast array of functional roles attributed to serotonin (5-HT) in the brain, changes in 5-HT levels have been shown to accompany changes in behavioral states, including WAKE, NREM and REM sleep. Whether 5-HT dynamics at shorter time scales can be seen to delineate substates within these larger brain states remains an open question. Here, we performed simultaneous recordings of extracellular 5-HT using a recently-developed G Protein-Coupled Receptor-Activation-Based 5-HT sensor (GRAB5-HT3.0) and local field potential (LFP) in the hippocampal CA1, which revealed the presence of prominent ultraslow (<0.05 Hz) 5-HT oscillations both during NREM and WAKE states. Interestingly, the phase of these ultraslow 5-HT oscillations was found to distinguish substates both within and across larger behavioral states. Hippocampal ripples occurred preferentially on the falling phase of ultraslow 5-HT oscillations during both NREM and WAKE, with higher power ripples concentrating near the peak specifically during NREM. By contrast, hippocampal-cortical coherence was strongest and microarousals and EMG peaks were most prevalent during the rising phase in both wake and NREM. Overall, ultraslow 5-HT oscillations delineate substates within the larger behavioral states of NREM and WAKE, thus potentially temporally segregating internal memory consolidation processes from arousal-related functions.

Chronic β3 adrenergic agonist treatment improves brain microvascular endothelial function and cognition in aged mice
Microvascular endothelial dysfunction, characterized by impaired neurovascular coupling, reduced glucose uptake, blood-brain barrier disruption, and microvascular rarefaction, plays a critical role in the pathogenesis of age-related vascular cognitive impairment (VCI). Emerging evidence points to non-cell autonomous mechanisms mediated by adverse circulating milieu (an increased ratio of pro-geronic to anti-geronic circulating factors) in the pathogenesis of endothelial dysfunction leading to impaired cerebral blood flow and cognitive decline in the aging population. In particular, age-related adipose dysfunction contributes, at least in part, to an unfavorable systemic milieu characterized by chronic hyperglycemia, hyperinsulinemia, dyslipidemia, and altered adipokine profile, which together contribute to microvascular endothelial dysfunction. Hence, in the present study, we aimed to test whether thermogenic stimulation, an intervention known to improve adipose and systemic metabolism by increasing cellular energy expenditure, could mitigate brain endothelial dysfunction and improve cognition in the aging population. Eighteen-month-old old C57BL/6J mice were treated with saline or CL ({beta}3-adrenergic agonist) for 6 weeks followed by functional analysis to assess endothelial function and cognition. CL treatment improved neurovascular coupling responses and rescued brain glucose uptake in aged animals. In addition, CL treatment also attenuated blood-brain barrier leakage and associated neuroinflammation in the cortex of aged animals. More importantly, these beneficial changes in microvascular function translated to improved cognitive performance in radial arm water maze and Y-maze tests. Our results suggest that {beta}3-adrenergic agonist treatment improves multiple aspects of brain microvascular endothelial function and can be potentially repurposed for treating age-associated cognitive decline.

Influence of asymmetric microchannels in the structure and function of engineered neuronal circuits
Understanding the intricate structure-function relationships of neuronal circuits is crucial for unraveling how the brain sustains efficient information transfer. In specific brain regions, like the hippocampus, neurons are organized in layers and form unidirectional connectivity, which is thought to help ensure controlled signal flow and information processing. In recent years, researchers have tried emulating these structural principles by providing cultured neurons with asymmetric environmental cues, namely microfluidics microchannels that promote directed axonal growth. Even though a few reports have claimed achieving unidirectional connectivity of in vitro neuronal circuits, given the lack of functional characterization, whether this structural connectivity correlates with functional connectivity remains unknown.

We have replicated and tested the performance of asymmetric microchannel designs previously reported in the literature to be successful in the promotion of directed axonal growth, as well as other custom variations. A new variation of "Arrowhead", termed "Rams", was the best-performing motif with a [~]76% probability per microchannel of allowing strictly unidirectional connections at 14 days in vitro. Importantly, we assessed the functional implications of these different asymmetric microchannel designs. For this purpose, we combined custom microfluidics with microelectrode array (MEA) technology to record the electrophysiological activity of two segregated populations of hippocampal neurons ("Source" and "Target"). This functional characterization revealed that up to [~]94% of the spiking activity recorded along microchannels with the "Rams" motif propagates towards the "Target" population. Moreover, our results indicate that these engineered circuits also tended to exhibit network-level synchronizations with defined directionality.

Overall, this characterization of the structure-function relationships promoted by asymmetric microchannels has the potential to provide insights into how neuronal circuits use specific network architectures for effective computations. Moreover, the here-developed devices and approaches may be used in a wide range of applications, such as disease modeling or preclinical drug screening.

Sulfonylurea receptor coupled conductances alter the performance of two central pattern generating circuits in Cancer borealis
Neuronal activity and energy supply must maintain a fine balance for neuronal fitness. Various channels of communication between the two could impact network output in different ways. Sulfonylurea receptors (SURs) are a modification of ATP-binding cassette proteins (ABCs) that confer ATP-dependent gating on their associated ion channels. They are widely expressed and link metabolic states directly to neuronal activity. The role they play varies in different circuits, both enabling bursting and inhibiting activity in pathological conditions. The crab, Cancer borealis, has central patterns generators (CPGs) that fire in rhythmic bursts nearly constantly and it is unknown how energy availability influences these networks. The pyloric network of the stomatogastric ganglion (STG) and cardiac ganglion (GC) control rhythmic contractions of the foregut and heart respectively. Pharmacological manipulation of SURs results in opposite effects in the two CPGs. Neuronal firing completely stops in the STG when SUR-associated channels are open, and firing increases when the channels are closed. This results from a decrease in the excitability of pyloric dilator (PD) neurons, which are a part of the pacemaker kernel. The neurons of the CG, paradoxically, increase firing within bursts when SUR-associated channels are opened, and bursting slows when SUR-associated channels are closed. The channel permeability and sensitivities analyses present novel SUR-conductance biophysics, which nevertheless change activity in ways reminiscent of the predominantly studied mammalian receptor/channels. We suggest that SUR-associated conductances allow different neurons to respond to energy states in different ways through a common mechanism.

Mitigating sTNF/TNFR1 activation on VGluT2+ spinal cord interneurons improves immune function after mid-thoracic spinal cord injury
Spinal cord injury (SCI) is a devastating condition with 250,000 to 500,000 new cases globally each year. Respiratory infections, e.g., pneumonia and influenza are the leading cause of death after SCI. Unfortunately, there is a poor understanding of how altered neuro-immune communication impacts an individuals outcome to infection. In humans and rodents, SCI leads to maladaptive changes in the spinal-sympathetic reflex (SSR) circuit which is crucial to sympathetic function. The cause of the impaired immune function may be related to harmful neuroinflammation which is detrimental to homeostatic neuronal function, aberrant plasticity, and hyperexcitable circuits. Soluble tumor necrosis factor (sTNF) is a pro-inflammatory cytokine that is elevated in the CNS after SCI and remains elevated for several months after injury. By pharmacologically attenuating sTNF in the CNS after SCI we were able to demonstrate improved immune function. Furthermore, when we investigated the specific cellular population which may be involved in altered neuro-immune communication we reported that excessive TNFR1 activity on excitatory INs promotes immune dysfunction. Furthermore, this observation is NF-kB dependent in VGluT2+ INs. Our data is the first report of a target within the CNS, TNFR1, that contributes to SCI-induced immune dysfunction after T9-SCI and is a potential avenue for future therapeutics.

Engineering Microglial Cells to Promote Spinal Cord Injury Recovery
Spinal cord injury (SCI) can result in irreversible damage, leading to lifelong paralysis for affected individuals. Microglias dual impact on neuronal regeneration after SCI, driven by their distinct roles at different stages, merits further study. We conducted a bioinformatic analysis of single-cell transcriptomes (scRNA), spatial transcriptomic (ST) data, and bulk RNA-seq data from Gene Expression Omnibus (GEO) datasets. The data were processed using R packages such as "Seurat", "DESeq2","limma" and "GSVA." Additionally, we utilized Gene Set Enrichment Analysis (GSEA) and the Enrichr web servers. Analysis of single-cell data and spatial transcriptomics has revealed notable changes in the microglial cell landscape in SCI. These changes encompass the inhibition of innate microglial cells, while reactive microglial cells exhibit pronounced reactive hyperplasia. Moreover, the TGF{beta} signaling pathway plays a crucial role in regulating the migration of innate microglial cells to enhance SCI recovery. However, reactive microglial cells exhibiting high Trem2 expression contribute to the neuroinflammatory response and can effectively modulate neural cell death in SCI. In particular, inhibiting Trem2 in reactive microglial cells not only reduces inflammation but also mitigates spinal cord injury, and enhancing the TGF{beta} signaling pathway. Whats more, the use of iPSC-derived microglial cells, which have demonstrated their capacity to augment the potential for replacing the functions of naive microglial cells, iPSC-derived microglia have the potential to replace the functions of naive microglial cells, holds significant promise in addressing SCI. Therefore, we posit that the engineering of microglial cells to promote the SCI recovery. The approach of inhibiting Trem2-mediated neuroinflammatory responses and transplanting iPSC-derived microglia with long-term TGF{beta} stimulation may offer potential improvements in SCI recovery.

Msh3 and Pms1 Set Neuronal CAG-repeat Migration Rate to Drive Selective Striatal and Cortical Pathogenesis in HD Mice
Modifiers of Huntingtons disease (HD) include mismatch repair (MMR) genes; however, their underlying disease-altering mechanisms remain unresolved. Knockout (KO) alleles for 9 HD GWAS modifiers/MMR genes were crossed to the Q140 Huntingtin (mHtt) knock-in mice to probe such mechanisms. Four KO mice strongly (Msh3 and Pms1) or moderately (Msh2 and Mlh1) rescue a triad of adult-onset, striatal medium-spiny-neuron (MSN)-selective phenotypes: somatic Htt DNA CAG-repeat expansion, transcriptionopathy, and mHtt protein aggregation. Comparatively, Q140 cortex also exhibits an analogous, but later-onset, pathogenic triad that is Msh3-dependent. Remarkably, Q140/homozygous Msh3-KO lacks mHtt aggregates in the brain, even at advanced ages (20-months). Moreover, Msh3-deficiency prevents striatal synaptic marker loss, astrogliosis, and locomotor impairment in HD mice. Purified Q140 MSN nuclei exhibit highly linear age-dependent mHtt DNA repeat expansion (i.e. repeat migration), with modal-CAG increasing at +8.8 repeats/month (R2=0.98). This linear rate is reduced to 2.3 and 0.3 repeats/month in Q140 with Msh3 heterozygous and homozygous alleles, respectively. Our study defines somatic Htt CAG-repeat thresholds below which there are no mHtt nuclear (at 150-CAG) or neuropil aggregates (at 192-CAG). Mild transcriptionopathy can still occur in Q140 mice with stabilized Htt 140-CAG repeats, but the majority of transcriptomic changes are due to somatic repeat expansion. Our analysis reveals 479 genes with expression levels highly correlated with modal-CAG length in MSNs. Thus, our study mechanistically connects HD GWAS genes to selective neuronal vulnerability in HD, in which Msh3 and Pms1 set the linear rate of neuronal mHtt CAG-repeat migration to drive repeat-length dependent pathogenesis; and provides a preclinical platform for targeting these genes for HD suppression across brain regions.

One Sentence SummaryMsh3 and Pms1 are genetic drivers of sequential striatal and cortical pathogenesis in Q140 mice by mediating selective CAG-repeat migration in HD vulnerable neurons.

HDAC4 Inhibits NMDA Receptor-Mediated Stimulation of Neurogranin Expression
The coordination of neuronal wiring and activity within the central nervous system (CNS) is crucial for cognitive function, particularly in the context of aging and neurological disorders. Neurogranin (Ng), an abundant forebrain protein, modulates calmodulin (CaM) activity and deeply influences synaptic plasticity and neuronal processing. This study investigates the regulatory mechanisms of Ng expression, a critical but underexplored area for combating cognitive impairment. Utilizing both in vitro and in vivo hippocampal models, we show that Ng expression arises during late developmental stages, coinciding with synaptic maturation and neuronal circuit consolidation of. We observed that Ng expression increases in neuronal networks with heightened synaptic activity and identified GluN2B-containing N-methyl-D-aspartate (NMDA) receptors as key drivers of this expression. Additionally, we discovered that nuclear-localized HDAC4 inhibits Ng expression, establishing a regulatory axis that is counteracted by NMDA receptor stimulation. Analysis of the Ng gene promoter activity revealed regulatory elements between the -2.4 and -0.85 Kbp region, including a binding site for RE1-Silencing Transcription factor (REST), which may mediate HDAC4s repressive effect on Ng expression. Further analysis of the promoter sequence revealed conserved binding sites for the myocyte enhancer factor-2 (MEF2) transcription factor, a target of HDAC4-mediated transcription regulation. Our findings elucidate the interplay between synaptic activity, NMDAR function, and transcriptional regulation in controlling Ng expression, offering insights into synaptic plasticity mechanisms and potential therapeutic strategies to prevent cognitive dysfunction.

Post-weaning social isolation alters sociability in a sex-specific manner
Adolescence is a critical period for brain development in humans and stress exposure during this time can have lasting effects on behavior and brain development. Social isolation and loneliness are particularly salient stressors that lead to detrimental mental health outcomes particularly in females, although most of the preclinical work on social isolation has been done in male animals. Our lab has developed a model of post-weaning adolescent social isolation that leads to increased drug reward sensitivity and altered neuronal structure in limbic brain regions. The current study utilized this model to determine the impact of adolescent social isolation on a three-chamber social interaction task both during adolescence and adulthood. We found that while post-weaning isolation does not alter social interaction during adolescence (PND45), it has sex-specific effects on social interaction in adulthood (PND60), potentiating social interaction in male mice and decreasing it in female mice. As early life stress can activate microglia leading to alterations in neuronal pruning, we next examined the impact of inhibiting microglial activation with daily minocycline administration during the first three weeks of social isolation on these changes in social interaction. During adolescence, minocycline dampened social interaction in male mice, while having no effect in females. In contrast, during adulthood, minocycline did not alter the impact of adolescent social isolation in males, with socially isolated males exhibiting higher levels of social interaction compared to their group housed counterparts. In females, adolescent minocycline treatment reversed the effect of social isolation leading to increased social interaction in the social isolation group, mimicking what is seen in naive males. Taken together, adolescent social isolation leads to sex-specific effects on social interaction in adulthood and adolescent minocycline treatment alters the effects of social isolation in females, but not males.

Enhancing Retrieval Capacity of the Predictive Brain through Dorsolateral Prefrontal Cortex Intervention
The ability to extract spatial or temporal regularities across experiences is crucial for skill development and predictive processes. The prefrontal cortex (PFC) plays a key role in modulating competitive memory systems, supporting declarative/episodic memory as opposed to statistical learning. This regulatory role may explain findings of improved acquisition and consolidation of statistical regularities following the suppression of dorsolateral PFC (DLPFC) by repetitive transcranial magnetic stimulation (rTMS). This raises a key question: Is access to models and prior statistical knowledge also modulated by the DLPFC? This preregistered study provides new insights by examining the role of the DLPFC in retrieving pre-existing knowledge of temporally distributed statistical regularities. Using a probabilistic learning task, healthy participants engaged in implicit statistical learning for 25 minutes. After a 24-hour consolidation period, participants received either 1 Hz rTMS or sham stimulation over the left, right, or bilateral DLPFC for 10 minutes before retesting. We found more effective access to statistical regularities in the bilateral DLPFC group compared to the sham group. Our results suggest that DLPFC suppression enhances the retrieval of statistical knowledge, particularly when interhemispheric compensatory mechanisms are prevented. These findings contribute to understanding competitive memory systems and offer implications for cognitive enhancement strategies.

Fundamental frequency predominantly drives talker differences in auditory brainstem responses to continuous speech
Deriving human neural responses to natural speech is now possible, but the responses to male- and female-uttered speech have been shown to differ. These talker differences may complicate interpretations or restrict experimental designs geared toward more realistic communication scenarios. This study found that when a male and female talker had the same fundamental frequency, auditory brainstem responses (ABRs) were very similar. Those responses became smaller and later with increasing fundamental frequency, as did click ABRs with increasing stimulus rates. Modeled responses suggested that the speech and click ABR differences were reasonably predicted by peripheral and brainstem processing of stimulus acoustics.

From histology to macroscale function in the human amygdala
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 ultrahigh 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 the human amygdala, describing inferior-superior as well as medio-lateral trends in microstructural differentiation that in part recapitulated established atlases of amygdala subnuclei. We then translated our approach to in vivo MRI data acquired at 7 Tesla, and could demonstrate 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 demonstrated 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.

Neuron synchronization analyzed through spatial-temporal attention
Across diverse organisms, the temporal dynamics of spiking responses between neurons, the neural synchrony, is crucial for encoding different stimuli. Neural synchrony is especially important in the insect antennal (olfactory) lobe (AL). Previous studies on synchronization, however, rely on pair-wise synchronization metrics including the cross-correlogram and cos-similarity between kernelized spikes train. These pair-wise analyses overlook an important aspect of synchronization which is the interaction at the population neuron level. There are also limited modeling techniques that incorporate the synchronization between neurons in modeling population spike trains. Inspired by recent advancements in machine learning, we leverage a modern attention mechanism to learn a generative normalizing flow that captures neuron population synchronization. Our method not only reveals the spiking mechanism of neurons in the AL region but also produces semi-interpretable attention weights that characterize neuron interactions over time. These automatically learned attention weights allow us to elucidate the known principles of neuron synchronization and further shed light on the functional roles of different cell types (the local interneurons (LNs), and projection neurons (PNs)) in the dynamic neural network in the AL. By varying the balance of excitation and inhibition in this neural circuit, our method further uncovers the pattern between the strength of synchronization and the ratio of an odorant in the mixture.

Author SummaryThe olfactory system can accurately compute the mixture of volatile compounds emitted from distant sources, enabling the foraging species to exhibit fast and effective decisions. However, altering ratios of one of the compounds in the mixture could be perceived as a different odor. Leveraging the current understanding of neural synchronization on sensory neural regions of insects, we construct a spatial-temporal attention normalizing flow, which partially replicates the AL regions functionality by learning the spiking mechanics of neurons. Beyond providing insights of the spiking mechanism of neurons in the AL region, our method also produces semi-interpretable attention weights that characterize neuron interaction over time. These automatically learned attention weights allow us to dissect out the principles of neuron synchronization and interaction mechanisms between projection neurons (PNs) and local neurons (LNs). Utilizing our accurate model of these AL functionality, we show evidence that the behavioral relevant compounds are closely clustered together while varying the intensities of one of the behavioral compounds in the mixture could attenuate the synchronization

Synchronous theta networks characterize successful memory retrieval
Memory retrieval activates regions across the brain, including not only the hippocampus and medial temporal lobe (MTL), but also frontal, parietal, and lateral temporal cortical regions. How these regions communicate to organize retrieval-specific processing, however, remains unclear. Here, we elucidate the role of theta (3-8 Hz) synchronization, broadly implicated in memory function, during the spontaneous retrieval of episodic memories. Analyzing a dataset of 413 neurosurgical patients implanted with intracranial electrodes who completed a free recall task, we find that synchronous networks of theta phase synchrony span the brain in the moments before spontaneous recall, in comparison to periods of deliberation and incorrect recalls. Network hubs, which systematically synchronize with other regions, appear throughout the prefrontal cortex and lateral and medial temporal lobes, as well as other areas. The recall accuracy network, derived from a correct recall-intrusion contrast, includes synchronous hubs concentrated in the temporal lobe and desynchronous hubs in the parietal lobe. Theta synchrony increases appear more prominently for slow (3 Hz) theta than for fast (8 Hz) theta in the recall-deliberation contrast, but not in the encoding or recall-intrusion contrast, and theta power and synchrony positively correlate throughout the theta band. These results implicate diffuse brain-wide synchronization of theta rhythms, especially slow theta, in episodic memory retrieval.

Significance StatementAnalyzing intracranial recordings from 413 subjects who completed an episodic free recall experiment, we analyze the brain-wide theta synchrony effects of memory retrieval. The literature has not previously described the whole-brain regional distribution of these effects nor studied them with respect to intrusions. We show that synchronous medial temporal hubs and desynchronous parietal hubs mark the recall accuracy network, and that theta synchrony in the successful encoding, successful retrieval, and recall accuracy contrasts correlates positively with theta power increases at a region. These findings significantly advance our understanding of the role and localization of theta synchrony effects during human memory retrieval.

Does pre-speech auditory modulation reflect processes related to feedback monitoring or speech movement planning?
Previous studies have revealed that auditory processing is modulated during the planning phase immediately prior to speech onset. To date, the functional relevance of this pre-speech auditory modulation (PSAM) remains unknown. Here, we investigated whether PSAM reflects neuronal processes that are associated with preparing auditory cortex for optimized feedback monitoring as reflected in online speech corrections. Combining electroencephalographic PSAM data from a previous data set with new acoustic measures of the same participants speech, we asked whether individual speakers extent of PSAM is correlated with the implementation of within-vowel articulatory adjustments during /b/-vowel-/d/ word productions. Online articulatory adjustments were quantified as the extent of change in inter-trial formant variability from vowel onset to vowel midpoint (a phenomenon known as centering). This approach allowed us to also consider inter-trial variability in formant production and its possible relation to PSAM at vowel onset and midpoint separately. Results showed that inter-trial formant variability was significantly smaller at vowel midpoint than at vowel onset. PSAM was not significantly correlated with this amount of change in variability as an index of within-vowel adjustments. Surprisingly, PSAM was negatively correlated with inter-trial formant variability not only in the middle but also at the very onset of the vowels. Thus, speakers with more PSAM produced formants that were already less variable at vowel onset. Findings suggest that PSAM may reflect processes that influence speech acoustics as early as vowel onset and, thus, that are directly involved in motor command preparation (feedforward control) rather than output monitoring (feedback control).