Biology subjects

Binshtok, A. M.

Publications and source records attributed to Binshtok, A. M..

10 recordsLinked to original sources

bioRxiv2026

Direct voltage imaging of peripheral axons in vivo reveals ongoing nociceptor firing in healthy mice

The electrical output of a neuron is generated and conducted along its axon, yet the axon has remained largely inaccessible to direct measurement of membrane potential in intact tissue. Genetically encoded voltage indicators have enabled optical recording of membrane potential dynamics from neuronal cell bodies and dendrites in vivo, but direct voltage imaging of identified axons in the living animal has remained largely unexplored. This gap is especially consequential for nociceptive neurons, whose signals are generated and propagated at peripheral terminals far from the soma. Here, we expressed genetically encoded voltage indicators in trigeminal neurons and, using high-speed widefield imaging with holographic targeted illumination, recorded membrane-voltage dynamics directly from corneal nociceptive terminal arbors and their parent axons in vivo, in intact tissue. Individual terminals responded to capsaicin and menthol with depolarization accompanied by trains of spiking activity. Unexpectedly, a substantial fraction generated ongoing action-potential-like firing in the absence of an applied stimulus. This firing was sodium-channel-dependent, synchronized across sister terminal branches, and shared its dynamics with the parent axon. Ongoing terminal firing persisted during lidocaine delivery to the trigeminal ganglion, supporting a peripheral origin distal to the ganglion, and was abolished by inhibition of NaV1.8 or HCN/Ih conductances. Thus, direct voltage imaging reveals ongoing and spatially coordinated electrical activity within intact peripheral sensory arbors. More broadly, this approach provides access to the generation and integration of neuronal output within fine axonal compartments in vivo.

neuroscience↗

bioRxiv2026

TRPV1-Mediated Delivery of Chloroprocaine, a Local Anesthetic with High pKa, Produces Pain-Selective Anesthesia Without Neurotoxicity

BackgroundConventional local anesthetics (LAs) provide pain relief but also block motor and non-nociceptive fibers, causing undesirable side effects. Approaches using permanently charged derivatives, such as QX-314, delivered via TRPV1 channels, selectively target nociceptors but are limited by neurotoxicity. We hypothesized that clinically approved LAs with high pKa and low toxicity, such as 2-chloroprocaine (chloroprocaine), which is predominantly protonated and membrane-impermeant at physiological pH, would preferentially enter TRPV1-expressing nociceptors when co-administered with a TRPV1 agonist, thereby producing prolonged pain-selective anesthesia without neurotoxicity. MethodsWhole-cell voltage-clamp recordings from rat dorsal root ganglion (DRG) neurons assessed sodium current inhibition. Behavioral experiments in male Sprague-Dawley rats (N=6 -12/group) evaluated sensitivity to noxious thermal and mechanical stimuli and motor function following intraplantar and perisciatic injections of 0.5% or 2% chloroprocaine combined with capsaicin or cannabidiol (CBD). TRPV1 activation was examined by calcium imaging in hTRPV1-expressing HEK293 cells and in capsaicin- and AITC-sensitive DRG neurons. Neurotoxicity was assessed using propidium iodide uptake, long-term behavioral monitoring (35 days), and immunohistochemistry for TRPV1, ATF-3, and GFAP. ResultsCo-application of chloroprocaine with capsaicin enhanced sodium current blockade (76{+/-}15%) compared to either drug alone (p<0.05). Perisciatic injection produced thermal analgesia that persisted for 4 hours, whereas motor block resolved within 30 minutes. Unlike other LAs, chloroprocaine did not activate TRPV1 (98% non-responders) and showed no cytotoxicity. Long-term evaluation revealed no hypersensitivity, neuronal loss, or ATF-3 upregulation. Replacing capsaicin with CBD preserved prolonged ([~] 3.5 h) pain-selective anesthesia without motor impairment. ConclusionsTRPV1-mediated delivery of high-pKa LA chloroprocaine produces prolonged, nociceptor-selective anesthesia without neurotoxicity. Combining a clinically approved high-pKa LAs with low intrinsic toxicity and TRPV1 agonists such as CBD offers a potentially safe strategy for pain-selective regional anesthesia, with implications for perioperative and chronic pain management.

neuroscience↗

bioRxiv2025

Prolactin Shapes Cortical Plasticity in Fathers

Caregiving alters the mammalian brain to support infant survival. While hypothalamic circuits are known to drive innate parental behaviors, information about the effect of parenthood on sensory processing and perception remain scarce, especially in fathers. We longitudinally imaged the same neurons in male mouse primary auditory cortex (ACx) before, during, and after fatherhood using two-photon calcium imaging. Behaviorally, males shifted from pup-directed aggression and self-oriented behaviors to transient parental care. In ACx, fathers showed enhanced and faster discrimination of pup ultrasonic vocalizations compared with matched narrowband noise. Slice electrophysiology revealed increased intrinsic excitability accompanies fatherhood. Mechanistically, we found elevated prolactin signaling in ACx, and manipulating prolactin levels modified the improvement in neuronal discriminability. Together, these results identify-in males-cortical plasticity and link prolactin to experience-dependent plasticity of sensory circuits that support caregiving. Our findings outline a father-specific mechanism that differs in key aspects from similar mechanisms in mothers.

neuroscience↗

bioRxiv2025

Efferent signaling along nociceptive peripheral terminals in vivo is enhanced during inflammation

Primary nociceptors are essentially characterized as afferent neurons carrying noxious sensory information from the periphery to the CNS. However, the information flow on primary nociceptors is bidirectional. Nociceptor peripheral terminals release a variety of mediators to the target organ in the vicinity of the injured area. These mediators promote sensitization of adjacent sensory neurons, vasodilation, and edema and affect innate and adaptive immunity, leading to hyperalgesia and inflammation that often expands beyond the injured areas. Many theories associate these phenomena with the antidromic action potential propagation along nociceptor terminals; however, the antidromic efferent signaling at the single nociceptor terminals has never been demonstrated. Here, using in vivo calcium imaging from the individual nociceptive terminals innervating the mouse cornea together with a computational approach, we demonstrated that short-lasting activation of a single terminal in vivo was sufficient to activate the remote, non-activated terminal, which branches from the same nociceptor fiber. This increase was dependent on the activation of voltage-gated sodium and calcium channels. Moreover, we showed that the efferent signaling along nociceptive terminals increases under inflammatory conditions, culminating in enhanced calcium signaling in the remote non-activated terminals. This inflammation-induced increase in intra-terminal calcium could trigger the enhanced release of inflammatory mediators, spilling over wider areas and affecting terminals from adjacent unstimulated receptive fields, leading to the expansion of hyperalgesia and inflammation.

neuroscience↗

bioRxiv2024

Adaptation of pain-related projection neurons in acute but not chronic pain

Pain hypersensitivity is associated with increased activity of peripheral and central neurons along the pain neuroaxis1. On the other hand, in other neuronal systems, increased activity leads to adaptive reduction of neuronal excitability to maintain homeostasis2-4. Projection neurons (PNs) of spinal and medullary dorsal horns summate the activity of primary nociceptive and local central interneurons and convey it to higher centers5. We show that at the peak of acute inflammatory pain, PNs reduce their intrinsic excitability and, consequently, action potential firing. When pain resolves, the excitability of PNs returns to baseline. Using electrophysiological and computational approaches, we found that an increase in potassium A-current (IA) underlies the decrease in the excitability of PNs in acute pain conditions. We hypothesized that an IA-induced decrease in PNs firing may restrain the output from the dorsal horn to prevent sensitization and pain chronification. Indeed, no changes of IA in PNs were observed in chronic pain conditions, and PNs exhibit increased intrinsic excitability and firing. Our results reveal an adaptive mechanism in acute pain conditions for regulating the output from the dorsal horn network, which, if interrupted, could trigger pain chronification.

neuroscience↗

bioRxiv2023

Age-dependent decrease in inhibitory drive on the excitatory superficial spinal dorsal horn neurons

The excitatory and inhibitory interneurons of superficial laminae I-II of the spinal dorsal horn (SDH) receive and process pain-related information from the primary afferents and transmit it to the brain via the projection neurons. Thus, the interaction between excitatory and inhibitory SDH interneurons is crucial in determining the output from the spinal cord network. Disruption of this interaction in pathological conditions leads to increased SDH output to the higher brain centers, which could underlie pathological pain. Here, we examined whether the changes in the intrinsic SDH connectivity also occur with age, possibly underlying age-related increase in pain sensitivity. Using Vgat;tdTomato transgenic mouse line, we compared the spontaneous inhibitory postsynaptic currents (sIPSCs) in inhibitory tdTomato+ and excitatory tdTomato- interneurons between adult (3-5 m.o.) and aged (12-13 m.o.) mice. We demonstrate that in adult mice, the amplitude and frequency of the sIPSCs in the excitatory interneurons were significantly higher than in inhibitory interneurons. These differences were annulled in aged mice. Further, we show that in aged mice, excitatory neurons receive less inhibition than in adult mice. This could lead to overall disinhibition of the SDH network, which might underlie increased pain perception among the aged population.

neuroscience↗

bioRxiv2023

Attenuation of colitis-induced visceral hypersensitivity and pain by silencing TRPV1-expressing fibers in rat colon

Background and AimsAbdominal pain in patients with inflammatory bowel disease (IBD) is common and debilitating. In our study, we aim to utilize transient receptor potential vanilloid 1 (TRPV1) channels, large-pore cation channels expressed on nociceptors, as a drug delivery system to selectively inhibit visceral nociceptors and thus visceral pain in a rodent model of IBD. MethodsWe induced colitis in rats using intrarectal dinitrobenzene sulfonic acid. Visceral hypersensitivity, spontaneous pain, and responsiveness of the hind paws to noxious heat stimuli were examined before and after the intrarectal application of sodium channel blocker QX-314 alone or together with TRPV1 channel activators or blockers. ResultsIntrarectal co-application of QX-314 with TRPV1 channel activator capsaicin significantly inhibited colitis-induced gut hypersensitivity. Furthermore, in the model of colitis, but not in naive rats, QX-314 alone was sufficient to reverse gut hypersensitivity. The blockade of TRPV1 channels prevented this effect of QX-314. Finally, applying QX-314 alone to the inflamed gut inhibited colitis-induced ongoing pain. ConclusionsSelective silencing of nociceptors by QX-314 entering via exogenously or endogenously activated TRPV1 channels diminish IBD-induced gut hypersensitivity. These results yet again confirm the central role of TRPV1-expressing nociceptive neurons in IBD pain. The lack of QX-314 effect on naive rats suggests its selective analgesic effect in IBD pain. Moreover, our results demonstrating the effect of QX-314 alone imply the role of a tonically active TRPV1 channel in the pathophysiology of IBD pain. This approach provides proof-of-concept for using charged activity blockers for selective and effective blockade of visceral pain.

neuroscience↗

bioRxiv2022

Structural plasticity of axon initial segment in spinal cord neurons underlies inflammatory pain

Activity-dependent structural plasticity of axon initial segment (AIS) regulates neuronal excitability, thus fine-tuning neuronal and overall network output. Here using behavioral, immunohistochemical, electrophysiological and computational approaches, we describe the structural plasticity of AIS in rats superficial spinal cord dorsal horn (SDH) neurons, which underlies inflammatory pain. We show an inflammation-mediated distal shift of the AIS away from the soma in inhibitory but not excitatory SDH neurons, concomitant with the peak of inflammatory pain. This AIS translocation was accompanied by a decrease in excitability of the inhibitory neurons. Following recovery from inflammatory hyperalgesia, the AIS location and neuronal excitability reversed to baseline levels. The computational model of SDH inhibitory neurons predicts that the distal shift of AIS is sufficient to decrease the intrinsic excitability of these neurons. Our results provide evidence of differential inflammation-mediated AIS plasticity, reducing the excitability of inhibitory but not excitatory SDH neurons and contributing to inflammatory hyperalgesia.

neuroscience↗

bioRxiv2021

Encoding of inflammatory hyperalgesia in mice spinal cord

Inflammation modifies the input-output properties of peripheral nociceptive neurons, thus leading to hyperalgesia, a condition in which the perception of noxious heat stimuli is altered such that the same stimulus produces enhanced pain. The increased nociceptive output enters the superficial dorsal spinal cord (SDH), which comprises the first CNS network integrating the noxious information. Here we used in vivo calcium imaging and a computational approach to investigate how the SDH network in mice encodes the injury-mediated abnormal input from peripheral nociceptive neurons. We show that the application of noxious heat stimuli to the hind paw in naive mice before induction of injury affects the activity of 70% of recorded neurons by either increasing or suppressing it. Application of the same noxious heat stimuli to hyperalgesic skin following injury leads to activation of previously non-responded cells and de-suppression of the "suppressed" neurons. We further demonstrate that reduction in synaptic inhibition mimics the response to the noxious stimuli in hyperalgesic conditions. Using a computational model of the SDH network, we predict that the "disinhibitory" effect of hyperalgesic stimuli results from the inflammation-mediated increased afferent input to the SDH network and a decrease in SDH inhibition. Both of these processes synergistically contribute to the injury-mediated increase in SDH output towards higher brain centers.

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bioRxiv2020

The input-output relation of primary nociceptive neurons is determined by the morphology of the peripheral nociceptive terminals

The output from the peripheral terminals of primary nociceptive neurons, which detect and encode the information regarding noxious stimuli, is crucial in determining pain sensation. The nociceptive terminal endings are morphologically complex structures assembled from multiple branches of different geometry, which converge in a variety of forms to create the terminal tree. The output of a single terminal is defined by the properties of the transducer channels producing the generation potentials and voltage-gated channels, translating the generation potentials into action potential firing. However, in the majority of cases, noxious stimuli activate multiple terminals; thus, the output of the nociceptive neuron is defined by the integration and computation of the inputs of the individual terminals. Here we used a computational model of nociceptive terminal tree to study how the architecture of the terminal tree affects input-output relation of the primary nociceptive neurons. We show that the input-output properties of the nociceptive neurons depend on the length, the axial resistance, and location of individual terminals. Moreover, we show that activation of multiple terminals by capsaicin-like current allows summation of the responses from individual terminals, thus leading to increased nociceptive output. Stimulation of terminals in simulated models of inflammatory or nociceptive hyperexcitability led to a change in the temporal pattern of action potential firing, emphasizing the role of temporal code in conveying key information about changes in nociceptive output in pathological conditions, leading to pain hypersensitivity. Significance statementNoxious stimuli are detected by terminal endings of the primary nociceptive neurons, which are organized into morphologically complex terminal trees. The information from multiple terminals is integrated along the terminal tree, computing the neuronal output, which propagates towards the CNS, thus shaping the pain sensation. Here we revealed that the structure of the nociceptive terminal tree determines the output of the nociceptive neurons. We show that the integration of noxious information depends on the morphology of the terminal trees and how this integration and, consequently, the neuronal output change under pathological conditions. Our findings help to predict how nociceptive neurons encode noxious stimuli and how this encoding changes in pathological conditions, leading to pain.

neuroscience↗