Publications using the Medusa

Diverse biological factors, such as sex and age, confer heterogeneity on sensory processing challenges in autism. These factors result in major difficulties in the processing of contextual information in social and non-social situations. To assess divergence in autistic traits, it is critical to consider sex- and age-related variability. Nevertheless, these differences remain largely elusive. Animal models of autism offer the possibility to examine contextual processing at the single-neuron level. Here, we investigated predictive processing of contextual auditory cues in the auditory midbrain of control and prenatally valproic acid-induced rats, a well-established animal model of autism. The rats were prepubertal and adult female and male animals. We performed single-unit recordings in the inferior colliculus of control and prenatally, or in utero , exposed rats under the classical oddball paradigm and non-repetitive cascade control sequences to study neuronal mismatch. This is the neuronal correlate of mismatch negativity, the brain’s automatic response to interruptions in environmental regularity. When comparing control and exposed rats, our results demonstrated a reduction in neuronal mismatch in rats exposed to valproic acid. However, exposed adult females exhibited an increased neuronal mismatch compared to their control counterparts. With respect to sex distinctions, valproic acid induced sex differences in neuronal mismatch of prepubertal and adult rats that are not observable in control animals. Moreover, we detected an age-dependent refinement in prediction error that is not affected by the drug. But valproic acid altered typical developmental trajectory of neuronal mismatch in both sexes. Such observations support sex- and age-related effects of in utero valproic acid exposure in contextual auditory processing at the neural level of the inferior colliculus. In autism, atypical predictive processing of environmental regularities underlies unusual responses to novel experiences. The present study highlights the importance of sex and age, that confer heterogeneity to these challenges.

Middle age represents a critical period of accelerated brain changes and provides a window for early detection and intervention in age-related neurological decline. Hearing loss is a key early marker of such decline and is linked to numerous comorbidities in older adults. Yet, ~10% of middle-aged individuals who report hearing difficulties show normal audiograms. Cochlear neural degeneration (CND) could contribute to these hidden hearing deficits, though its role remains unclear due to a lack of objective diagnostics and uncertainty regarding its perceptual outcomes. Here, we employed a cross-species design to examine neural and behavioral signatures of CND. We measured envelope following responses (EFRs) – neural ensemble responses to sound originating from the peripheral auditory pathway – in young and middle-aged adults with normal audiograms and compared these responses to young and middle-aged Mongolian gerbils, where CND was histologically confirmed. We observed near-identical changes in EFRs across species that were associated with CND. Behavioral assessments revealed age-related speech-in-noise deficits under challenging conditions, while pupil-indexed listening effort increased with age even when behavioral performance was matched. Together, these results demonstrate that CND contributes to speech perception difficulties and elevated listening effort in midlife, which may ultimately lead to listening fatigue and social withdrawal.

Intracortical microelectrodes are used for recording activity from individual neurons, providing both a valuable neuroscience tool and an enabling medical technology for individuals with motor disabilities. Standard neural probes carrying the microelectrodes are rigid silicon-based structures that can penetrate the brain parenchyma to interface with the targeted neurons. Unfortunately, within weeks after implantation, neural recording quality from microelectrodes degrades, owing largely to a neuroinflammatory response. Key contributors to the neuroinflammatory response include mechanical mismatch at the device-tissue interface and oxidative stress. We developed a mechanically-adaptive, resveratrol-eluting (MARE) neural probe to mitigate both mechanical mismatch and oxidative stress and thereby promote improved neural recording quality and longevity. In this work, we demonstrate that compared to rigid silicon controls, highly-flexible MARE probes exhibit improved recording performance, more stable impedance, and a healing tissue response. With further optimization, MARE probes can serve as long-term, robust neural probes for brain-machine interface applications.

Epidural electrical stimulation of the spinal cord (ES) is used to restore/improve locomotor movements in patients. However, the neuronal mechanisms underlying activation of locomotor networks by ES are unknown. Here, we analyzed the effects of ES on the activity of individual spinal neurons of different functional locomotor groups. Neuronal activity was recorded in decerebrate cats before and during ES that evoked locomotion. During ES initial period (NW-period, before the locomotion onset), the activity was increased in most neurons as compared to that before ES. We found that ES-caused activation of neurons of locomotor network to the average level similar to that observed during locomotion is not sufficient for initiation of locomotion. We demonstrated that the onset of ES-evoked locomotion was associated with specific changes in the activity of individual neurons within functional locomotor groups and in their responses to epidural stimuli. With the locomotion onset, there was a trend for individual neurons with extremely high activity during NW-period to decrease their activity, while for neurons with extremely low activity to become more active. Also, in neurons contributing to generation of a specific mode of locomotion, peaks and troughs in responses to individual epidural stimuli were significantly less pronounced as compared with those observed during NW-period. We suggest that these changes allow ES to maintain the high level of excitability of the locomotor network necessary for its operation without distortion of the locomotor rhythm. The obtained results advance our understanding of the neuronal mechanisms underlying activation of locomotor network by ES.

Presbycusis or age-related hearing loss (ARHL) affects millions of people worldwide, increasing their risk of cognitive decline and poor quality of life. However, ARHL remains an irreversible condition due to our inability to induce inner-ear hair cell regeneration. Nevertheless, multisession epidural stimulation of the auditory cortex (AC) at the onset of ARHL prevents hearing threshold elevation in naturally aging Wistar rats. Accordingly, we hypothesized that anodal direct current (DC) stimulation of the AC may also compensate for age-related maladaptive, activity-dependent changes. Here, we examined immunocytochemical markers in the AC, including early genes (c-fos and Arc), AMPA receptors (GluR2/3), parvalbumin (PV), and GAD67, along with auditory-evoked potentials (CAEPs) recorded in both auditory and visual (VC) cortices. When comparing 6 and 18.13-month-old rats without AC simulation, we observed loss of c-fos and Arc-positive neurons and decreased GluR2/3 expression, confirming altered AC neuronal network plasticity and activation. In addition, we noted changes in PV and decreased GAD67 immunoreactivity suggesting disrupted inhibition and significantly increased wave amplitudes in CAEPs, altered AC latencies, and decreased VC responses. By contrast, electrically stimulated rats showed no significant variations in early gene markers, GluR2/3, PV, or GAD67 with age, and the amplitudes and latencies of CAEPs recorded in their AC and VC resembled those of young rat. These findings indicate that anodal DC stimulation at the onset of ARHL delays AC aging by minimizing the loss of inhibition and preventing increases in cortical excitability in Wistar rats.

Epilepsy is a multifaceted neurological disorder characterized by recurrent seizures and associated with molecular and immune alterations in key brain regions. The GASH/Sal (Genetic Audiogenic Seizure Hamster, Salamanca), a genetic model for audiogenic epilepsy, provides a powerful tool to study seizure mechanisms and resistance in predisposed individuals. This study investigates the proteomic and immune responses triggered by audiogenic kindling in the inferior colliculus, comparing non-responder animals exhibiting reduced seizure severity following repeated stimulation versus GASH/Sal naïve hamsters. To assess auditory pathway functionality, Auditory Brainstem Responses (ABRs) were recorded, revealing reduced neuronal activity in the auditory nerve of non-responders, while central auditory processing remained unaffected. Cytokine profiling demonstrated increased levels of proinflammatory markers, including IL-1 alpha (Interleukin-1 alpha), IL-10 (Interleukin-10), and TGF-beta (Transforming Growth Factor beta), alongside decreased IGF-1 (Insulin-like Growth Factor 1) levels, highlighting systemic inflammation and its interplay with neuroprotection. Building on these findings, a proteomic analysis identified 159 differentially expressed proteins (DEPs). Additionally, bioinformatic approaches, including Gene Set Enrichment Analysis (GSEA) and Weighted Gene Co-expression Network Analysis (WGCNA), revealed disrupted pathways related to metabolic and inflammatory epileptic processes and a module potentially linked to a rise in the threshold of seizures, respectively. Differentially expressed genes, identified through bioinformatic and statistical analyses, were validated by RT-qPCR. This confirmed the upregulation of six genes (Gpc1—Glypican-1; Sdc3—Syndecan-3; Vgf—Nerve Growth Factor Inducible; Cpne5—Copine 5; Agap2—Arf-GAP with GTPase domain, ANK repeat, and PH domain-containing protein 2; and Dpp8—Dipeptidyl Peptidase 8) and the downregulation of two (Ralb—RAS-like proto-oncogene B—and S100b—S100 calcium-binding protein B), aligning with reduced seizure severity. This study may uncover key proteomic and immune mechanisms underlying seizure susceptibility, providing possible novel therapeutic targets for refractory epilepsy.

Humans and mice with mutations in genes encoding CIB2 and whirlin (WHRN) are deaf. We previously reported that CIB2 binds to WHRN and is essential for stereocilia staircase architecture of cochlear hair cells. Here, we refine the interaction domains of both proteins and show that these proteins play unique roles in stereocilia bundle formation and organization. We found that the EF2 domain of CIB2 binds to the HHD2 region of WHRN. AlphaFold2 multimer independently identified the same interacting regions and gave a thorough structural model. Next, we investigated genetic interaction between murine Cib2 and Whrn. Hearing in mice double heterozygous for functionally null alleles (Cib2KO/+;Whrnwi/+) was similar to that in age-matched wild-type mice, indicating that partial deficiency for both Cib2 and Whrn does not impair hearing. Double homozygous mutant mice (Cib2KO/KO;Whrnwi/wi) were deaf, and their cochlear stereocilia exhibited a predominant phenotype seen in single Whrnwi/wi mutants. Overexpression of WHRN in Cib2KO/KO mice did not rescue the stereocilia morphology. These data suggest that CIB2 is multifunctional, with key independent functions in the development and/or maintenance of the stereocilia staircase pattern in auditory hair cells.

It is crucial to manage hearing loss and its associated public health impacts. In this study, we aimed to understand the role of Sema3f in the development and maintenance of the auditory system. Inner ear-specific Sema3f knockout mice exhibited hearing loss at 8 weeks with an elevated threshold for auditory brainstem response and an absent threshold for distortion product optoacoustic emission tests. Additionally, an increased number of outer hair cells and abnormal patterns of spiral ganglion neuron projections in the outer hair cell regions were observed. Through the analyses of sequencing data from 558 families with hearing loss, we identified biallelic variants of SEMA3F, which encodes semaphorin-3F, in one of the families. In the family, the proband showed profound progressive nonsyndromic hearing loss with congenital onset. In vitro analysis revealed that the identified missense variants decreased the furin-mediated processing of SEMA3F and abolished the cellular abilities of SEMA3F, which collapsed the filamentous actin cytoskeleton in human umbilical vein-derived endothelial cells. Our data suggest that SEMA3F is essential for normal hearing and is associated with nonsyndromic hearing loss in humans.

Closed-loop neural control is a powerful tool for both the scientific exploration of neural function and for mitigating deficiencies found in open-loop deep brain stimulation (DBS). Here, we present a protocol for artificial intelligence-guided neural control in rats using deep reinforcement learning (RL) and infrared neural stimulation (INS). We describe steps for integrating RL closed-loop control into neuroscience and neuromodulation studies. We then detail procedures for using and deploying infrared INS in chronic DBS applications. For complete details on the use and execution of this protocol, please refer to Coventry et al.1 and Coventry and Bartlett.2.

CRISPR-Cas13 systems have therapeutic promise for the precise correction of point mutations in RNA. Using adenosine deaminase acting on RNA (ADAR) effectors, A-I base conversions can be targeted using guide RNAs (gRNAs). We compare the Cas13 effectors PspCas13b and Cas13bt3 for the repair of the gene USH2A , a common cause of inherited retinal disease and Usher syndrome. In cultured cells, we demonstrate up to 80% efficiency for the repair of the common c.11864 G > A and its murine equivalent c.11840 G > A, across different gRNAs and promoters. We develop and characterize a mouse model of Usher syndrome carrying the c.11840 G > A mutation designed for the evaluation of base editors for inherited retinal disease. Finally, we compare Cas13 effectors delivered via AAV for the repair of Ush2a in photoreceptors. Mean RNA editing rates in photoreceptors across different constructs ranged from 0.32% to 2.04%, with greater efficiency in those injected with PspCas13b compared to Cas13bt3 constructs. In mice injected with PspCas13b constructs, usherin protein was successfully restored and correctly localized to the connecting cilium following RNA editing. These results support the development of transcriptome targeting gene editing therapies for retinal disease.

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here, we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

This study involves longitudinal neuro-electrophysiological analysis using motor-evoked potentials (MEP) and the Basso, Beattie, and Bresnahan behavioral examinations (BBB) to evaluate moderate mid-thoracic contusive spinal cord injury (SCI) in a rat model. Objectives/Background: The objective of the study is to characterize the onset and progression of contusive SCI over an eight-week period using a clinically applicable tool in an in vivo model. The background highlights the importance of a reliable and reproducible injury model and assessment tools for SCI. Methods: The methods section describes the experimental setup, including randomly assigned rats in three groups: Sham, Control, and Injury (undergoing a moderate contusive SCI using the NYU-Impactor). MEP monitoring and BBB examinations are conducted at baseline and weekly for eight weeks post-injury. Results: The results indicate that the relative MEP power spectral decreased to 11% and 22% in the left and right hindlimbs, respectively, during the first week post-SCI. In the second week, a slight spontaneous recovery was observed, reaching 17% in the left and 31% in the right hindlimbs. Over the following four weeks post-SCI, continuing deterioration of MEP signal power was observed with no detectable recovery. Conclusions: SCI attenuates hindlimb MEP power spectral and reduces locomotion, though the changes in MEP and locomotion exhibit distinct temporal patterns. The MEP monitoring provides valuable insights into the functional integrity of motor pathways following SCI and offer a sensitive and reliable assessment. By implementing MEP monitoring, researchers can track the progression of SCI and evaluate the efficacy of therapeutic interventions quantitatively.

Hearing mediates many behaviours critical for survival in echolocating bats, including foraging and navigation. Although most mammals are susceptible to progressive age-related hearing loss, the evolution of biosonar, which requires the ability to hear low-intensity echoes from outgoing sonar signals, may have selected against the development of hearing deficits in bats. Many echolocating bats exhibit exceptional longevity and rely on acoustic behaviours for survival to old age; however, relatively little is known about the ageing bat auditory system. In this study, we used DNA methylation to estimate the ages of wild-caught big brown bats ( Eptesicus fuscus ) and measured hearing sensitivity in young and ageing bats using auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). We found no evidence for hearing deficits in bats up to 12.5 years of age, demonstrated by comparable thresholds and similar ABR and DPOAE amplitudes across age groups. We additionally found no significant histological evidence for cochlear ageing, with similar hair cell counts, afferent and efferent innervation patterns in young and ageing bats. Here, we demonstrate that big brown bats show minimal evidence for age-related hearing loss and therefore represent informative models for investigating mechanisms that may preserve hearing function over a long lifetime.

Myh1 is a mouse deafness gene with an unknown function in the auditory system. Hearing loss in Myh1 -knockout mice is characterized by an elevated threshold for the auditory brainstem response and the absence of a threshold for distortion product otoacoustic emission. Here, we investigated the role of MYH1 in outer hair cells (OHCs), crucial structures in the organ of Corti responsible for regulating cochlear amplification. Direct whole-cell voltage-clamp recordings of OHCs revealed that prestin activity was lower in Myh1 -knockout mice than in wild-type mice, indicating abnormal OHC electromotility. We analyzed whole-exome sequencing data from 437 patients with hearing loss of unknown genetic causes and identified biallelic missense variants of MYH1 in five unrelated families. Hearing loss in individuals harboring biallelic MYH1 variants was non-progressive, with an onset ranging from congenital to childhood. Three of five individuals with MYH1 variants displayed osteopenia. Structural prediction by AlphaFold2 followed by molecular dynamic simulations revealed that the identified variants presented structural abnormalities compared with wild-type MYH1. In a heterogeneous overexpression system, MYH1 variants, particularly those in the head domain, abolished MYH1 functions, such as by increasing prestin activity and modulating the membrane traction force. Overall, our findings suggest an essential function of MYH1 in OHCs, as observed in Myh1-deficient mice, and provide genetic evidence linking biallelic MYH1 variants to autosomal recessive hearing loss in humans.

Cochlear ribbon synapses between sensory inner hair cells (IHCs) and spiral ganglion neurons (SGNs) are vulnerable to rapid and primary damage and/or loss due to noise overexposure. Such damaged ribbon synapses can repair spontaneously in mouse and guinea pig. However, the mechanisms for synaptic repair are unclear. Previously, we have demonstrated a critical role for the fractalkine signaling axis (CX 3 CL1-CX 3 CR1) in synaptic repair, wherein noise-damaged ribbon synapses are spontaneously repaired in the presence of fractalkine receptor (CX 3 CR1) expressed by cochlear macrophages. Here, we examined whether local administration of chemokine fractalkine ligand (CX 3 CL1 or FKN) in the form of a peptide is effective in restoring synapses and hearing loss after noise-induced cochlear synaptopathy (NICS). Specifically, the efficacy of different isoforms of FKN was evaluated for restoration of loss of IHC ribbon synapses and hearing after NICS. A single transtympanic injection of soluble isoform of FKN (sFKN) peptide at 1 day after synaptopathic noise trauma for 2 hours at 93 decibel sound pressure level led to significant recovery of auditory brainstem response (ABR) thresholds, ABR peak I amplitudes and ribbon synapses in FKN knockout mice when compared to mice injected with membrane-bound FKN peptide (mFKN). Likewise, local treatment with sFKN peptide in FKN wild type mice restored synaptopathic noise-damaged ribbon synapses and ABR peak I amplitudes. Mechanistically, FKN regulates macrophage numbers in the damaged cochlea and in the absence of macrophages, sFKN failed to restore loss of synapses and hearing after NICS. Furthermore, sFKN treatment attenuated cochlear inflammation after NICS without altering the expression of CX 3 CR1. Finally, injected sFKN peptide was detectable inside the cochlea for 24 h localized to the basilar membrane and spiral lamina near the sensory epithelium. These data provide a proof-of-principle that local delivery of an immune factor, sFKN is effective in restoring ribbon synapses and hearing loss after NICS in a macrophage-dependent manner and highlights the potential of sFKN as an immunotherapy for cochlear synaptopathy due to noise.

Neural circuits in the auditory brainstem compute interaural time and intensity differences used to determine the locations of sound sources. These circuits display features that are specialized for these functions. The projection from the ventral cochlear nucleus (VCN) to the medial nucleus of the trapezoid (MNTB) body travels along highly myelinated fibers and terminates in the calyx of Held. This monoinnervating synapse emerges during development as multiple inputs are eliminated. We previously demonstrated that elimination of microglia with a colony stimulating factor-1 inhibitor results in impaired synaptic pruning so that multiple calyceal terminals reside on principal cells of MNTB. This inhibitor also resulted in impaired auditory brainstem responses (ABRs), with elevated thresholds and increased peak latencies. Loss of the microglial fractalkine receptor, CX3CR1, decreased peak latencies in the ABR. The mechanisms underlying these effects are not known. One prominent microglial signaling pathway involved in synaptic pruning and plasticity during development and aging is the C1q-initiated compliment cascade. Here we investigated the classical complement pathway initiator, C1q, in auditory brainstem maturation. We found that C1q expression is detected in the MNTB by the first postnatal week. C1q levels increased with age and were detected within microglia and surrounding the soma of MNTB principal neurons. Loss of C1q did not affect microglia-dependent calyceal pruning. Excitatory and inhibitory synaptic markers in the MNTB and LSO were not altered with C1q deletion. ABRs showed that C1q KO mice had normal hearing thresholds but shortened peak latencies. Altogether this study uncovers the developmental time frame of C1q expression in the sound localization pathway and shows a subtle functional consequence of C1q knockdown.

Hair cells of the inner ear and lateral-line system rely on specialized ribbon synapses to transmit sensory information to the central nervous system. The molecules required to assemble these synapses are not fully understood. We show that Nrxn3, a presynaptic adhesion molecule, is crucial for ribbon-synapse maturation in hair cells. In both mouse and zebrafish models, the loss of Nrxn3 results in significantly fewer intact ribbon synapses. We show in zebrafish that, initially, Nrxn3 loss does not alter pre- and postsynapse numbers but, later, synapses fail to pair, leading to postsynapse loss. We also demonstrate that Nrxn3 subtly influences synapse selectivity in zebrafish lateral-line hair cells that detect anterior flow. Loss of Nrxn3 leads to a 60% loss of synapses in zebrafish, which dramatically reduces pre- and postsynaptic responses. Despite fewer synapses, auditory responses in zebrafish and mice are unaffected. This work demonstrates that Nrxn3 is a crucial and conserved molecule required for the maturation of ribbon synapses. Understanding how ribbon synapses mature is essential to generating new therapies to treat synaptopathies linked to auditory or vestibular dysfunction.

Hair cell bundles consist of stereocilia arranged in rows of increasing heights, connected by tip links that transmit sound-induced forces to shorter stereocilia tips. Auditory mechanotransduction channel complexes, composed of proteins TMC1/2, TMIE, CIB2, and LHFPL5, are located at the tips of shorter stereocilia. While most components can interact with the tip link in vitro, their ability to maintain the channel complexes at the tip link in vivo is uncertain. Return, using mouse models, we show that an additional component, LOXHD1, is essential for keeping TMC1-pore forming subunits at the tip link but is dispensable for TMC2. Using SUB-immunogold-SEM, we showed that TMC1 localizes near the tip link but mislocalizes without LOXHD1. LOXHD1 selectively interacts with TMC1, CIB2, LHFPL5, and tip-link protein PCDH15. Our results demonstrate that TMC1-driven mature auditory channels require LOXHD1 to stay connected to the tip link and remain functional, while TMC2-driven developmental channels do not.

Background Prepulse inhibition (PPI) is a phenomenon where a weak prepulse stimulus inhibits the startle reflex to a subsequent stronger stimulus, which can be induced by various sensory stimulus modalities such as visual, tactile, and auditory stimuli. Methods This study investigates the neural mechanisms underlying auditory PPI by focusing on the deep layers of the superior colliculus (deepSC) and the inferior colliculus (IC) in rats. Nineteen male Sprague-Dawley rats were implanted with electrodes in the left deepSC and the right IC, and electrophysiological recordings were conducted under anesthesia to observe the frequency following responses (FFRs) to startle stimuli with and without prepulse stimuli. Results Our results showed that in the deepSC, narrowband noise as a prepulse stimulus significantly inhibited the envelope component of the startle response, while the fine structure component remained unaffected. However, this inhibitory effect was not observed in the IC or when the prepulse stimulus was a gap. Conclusion These findings suggest that the deepSC plays a crucial role in the neural circuitry of PPI, particularly in the modulation of the envelope component of the startle response. The differential effects of narrowband noise and gap as prepulse stimuli also indicate distinct neural pathways for sound-induced PPI and Gap-PPI. Understanding these mechanisms could provide insights into sensory processing and potential therapeutic targets for disorders involving impaired PPI, such as tinnitus.

Background Spinal cord stimulation (SCS) has demonstrated multiple benefits in treating chronic pain and other clinical disorders related to sensorimotor dysfunctions. However, the underlying mechanisms are still not fully understood, including how electrode placement in relation to the spinal cord neuroanatomy influences epidural spinal recordings (ESRs). To characterize this relationship, this study utilized stimulation applied at various anatomical sections of the spinal column, including at levels of the intervertebral disc and regions correlating to the dorsal root entry zone. Method Two electrode arrays were surgically implanted into the dorsal epidural space of the swine. The stimulation leads were positioned such that the caudal-most electrode contact was at the level of a thoracic intervertebral segment. Intraoperative cone beam computed tomography (CBCT) images were utilized to precisely determine the location of the epidural leads relative to the spinal column. High-resolution microCT imaging and 3D-model reconstructions of the explanted spinal cord illustrated precise positioning and dimensions of the epidural leads in relation to the surrounding neuroanatomy, including the spinal rootlets of the dorsal and ventral columns of the spinal cord. In a separate swine cohort, implanted epidural leads were used for SCS and recording evoked ESRs. Results Reconstructed 3D-models of the swine spinal cord with epidural lead implants demonstrated considerable distinctions in the dimensions of a single electrode contact on a standard industry epidural stimulation lead compared to dorsal rootlets at the dorsal root entry zone (DREZ). At the intervertebral segment, it was observed that a single electrode contact may cover 20-25% of the DREZ if positioned laterally. Electrode contacts were estimated to be ~0.75 mm from the margins of the DREZ when placed at the midline. Furthermore, ventral rootlets were observed to travel in proximity and parallel to dorsal rootlets at this level prior to separation into their respective sides of the spinal cord. Cathodic stimulation at the level of the intervertebral disc, compared to an ‘off-disc’ stimulation (7 mm rostral), demonstrated considerable variations in the features of recorded ESRs, such as amplitude and shape, and evoked unintended motor activation at lower stimulation thresholds. This substantial change may be due to the influence of nearby ventral roots. To further illustrate the influence of rootlet activation vs. dorsal column activation, the stimulation lead was displaced laterally at ~2.88 mm from the midline, resulting in variances in both evoked compound action potential (ECAP) components and electromyography (EMG) components in ESRs at lower stimulation thresholds. Conclusion The results of this study suggest that the ECAP and EMG components of recorded ESRs can vary depending on small differences in the location of the stimulating electrodes within the spinal anatomy, such as at the level of the intervertebral segment. Furthermore, the effects of sub-centimeter lateral displacement of the stimulation lead from the midline, leading to significant changes in electrophysiological metrics. The results of this pilot study reveal the importance of the small displacement of the electrodes that can cause significant changes to evoked responses SCS. These results may provide further valuable insights into the underlying mechanisms and assist in optimizing future SCS-related applications.