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Skeletal muscle contraction triggers the exercise pressor reflex (EPR) to regulate the cardiovascular system response to exercise. During muscle contraction, substances are released that generate action potential activity in group III and IV afferents that mediate the EPR. Some of these substances increase afferent activity via G-protein-coupled receptor (GPCR) activation, but the mechanisms are incompletely understood. We were interested in determining if tetrodotoxin-resistant (TTX-R) voltage-dependent sodium channels (NasubV/sub) were involved and investigated the effect of a mixture of such compounds (bradykinin, prostaglandin, norepinephrine, and ATP, called muscle metabolites). Using whole cell patch-clamp electrophysiology, we show that the muscle metabolites significantly increased TTX-R NasubV/subcurrents. The rise time of this enhancement averaged ∼2 min, which suggests the involvement of a diffusible second messenger pathway. The effect of muscle metabolites on the current-voltage relationship, channel activation and inactivation kinetics support NasubV/sub1.9 channels as the target for this enhancement. When applied individually at the concentration used in the mixture, only prostaglandin and bradykinin significantly enhanced NasubV/subcurrent, but the sum of these enhancements waslt;1/3 that observed when the muscle metabolites were applied together. This suggests synergism between the activated GPCRs to enhance NasubV/sub1.9 current. When applied at a higher concentration, all four substances could enhance the current, which demonstrates that the GPCRs activated by each metabolite can enhance channel activity. The enhancement of NasubV/sub1.9 channel activity is a likely mechanism by which GPCR activation increases action potential activity in afferents generating the EPR.bNEWamp; NOTEWORTHY/bG-protein-coupled receptor (GPCR) activation increases action potential activity in muscle afferents to produce the exercise pressor reflex (EPR), but the mechanisms are incompletely understood. We provide evidence that NasubV/sub1.9 current is synergistically enhanced by application of a mixture of metabolites potentially released during muscle contraction. The enhancement of NasubV/sub1.9 current is likely one mechanism by which GPCR activation generates the EPR and the inappropriate activation of the EPR in patients with cardiovascular disease.

Vagal reflexes regulate homeostasis in visceral organs and systems through afferent and efferent neurons and nerve fibers. Small, unmyelinated, C-type afferents comprise over 80% of fibers in the vagus and form the sensory arc of autonomic reflexes of the gut, lungs, heart and vessels and the immune system. Selective bioelectronic activation of C-afferents could be used to mechanistically study and treat diseases of peripheral organs in which vagal reflexes are involved, but it has not been achieved.We stimulated the vagus in rats and mice using trains of kHz-frequency stimuli. Stimulation effects were assessed using neuronal c-Fos expression, physiological and nerve fiber responses, optogenetic and computational methods.Intermittent kHz stimulation for 30 min activates specific motor and, preferentially, sensory vagus neurons in the brainstem. At sufficiently high frequencies (5 kHz) and at intensities within a specific range (7-10 times activation threshold, T, in rats; 15-25 × T in mice), C-afferents are activated, whereas larger, A- and B-fibers, are blocked. This was determined by measuring fiber-specific acute physiological responses to kHz stimulus trains, and by assessing fiber excitability around kHz stimulus trains through compound action potentials evoked by probing pulses. Aspects of selective activation of C-afferents are explained in computational models of nerve fibers by how fiber size and myelin shape the response of sodium channels to kHz-frequency stimuli.kHz stimulation is a neuromodulation strategy to robustly and selectively activate vagal C-afferents implicated in physiological homeostasis and disease, over larger vagal fibers.

Enteroendocrine cells (EECs) are the primary sensory cells that sense the gut luminal environment and secret hormones to regulate organ function. Recent studies revealed that vagal afferent neurons are connected to EECs and relay sensory information from EECs to the brain stem. To date, however, the identity of vagal afferent neurons connected to a given EEC subtype and the mode of their gene responses to its intestinal hormone have remained unknown. Hypothesizing that EEC-associated vagal afferent neurons change their gene expression in response to the microbiota-related extracellular stimuli, we conducted comparative gene expression analyses of the nodose-petrosal ganglion complex (NPG) using specific pathogen-free (SPF) and germ-free (GF) mice. We report here that the Uts2b gene, which encodes a functionally unknown neuropeptide, urotensin 2B (UTS2B), is expressed in a microbiota-dependent manner in NPG neurons. In cultured NPG neurons, expression of Uts2b was induced by AR420626, the selective agonist for FFAR3. Moreover, distinct gastrointestinal hormones exerted differential effects on Uts2b expression in NPG neurons, where cholecystokinin (CCK) significantly increased its expression. The majority of Uts2b-expressing NPG neurons expressed CCK-A, the receptor for CCK, which comprised approximately 25% of all CCK-A-expressing NPG neurons. Selective fluorescent labeling of Uts2b-expressing NPG neurons revealed a direct contact of their nerve fibers to CCK-expressing EECs. This study identifies the Uts2b as a microbiota-regulated gene, demonstrates that Uts2b-expressing vagal afferent neurons transduce sensory information from CCK-expressing EECs to the brain, and suggests potential involvement of UTS2B in a modality of CCK actions.

Dental pain is a persistent, detrimental public health issue that requires a better understanding of the mechanisms of tooth pain and inflammation in order to develop more effective treatments. Calcitonin gene-related peptide (CGRP) and dental pulp cells are promising candidates for mediating tooth pain and generating reparative dental tissues, respectively, but their behavior in the context of pulpitis remains elusive. The mouse incisor requires Sonic hedgehog (Shh) secreted from sensory nerves to continuously regenerate. However, it is unknown whether sensory nerves also regulate the comparatively nonregenerative mouse molar through CGRP and Shh. This is an important knowledge gap to fill since mouse incisors differ biologically from human teeth, while mouse and human molars are similar. In this work, we identified that molar pulp cells express CGRP receptor and Gli1, a Hedgehog (Hh) signaling protein found to label a dental stem cell population in the mouse incisor. We also observed in a mouse molar injury model that Hh signaling was activated and Shh expression was upregulated in vivo. We then determined in vitro that Shh and CGRP regulate differentiation of primary mouse molar and incisor pulp cells and a human dental pulp stem cell line. Furthermore, conditioned media from stimulated sensory neurons induced Hh signaling activation and inflammatory gene expression in primary molar pulp cells, which was abolished by inhibition of either Shh or CGRP. Our results suggest that CGRP and Shh signaling may promote an inflammatory response after injury in the molar and that activated sensory nerves secrete CGRP and Shh to regulate molar pulp cell expansion and differentiation into odontoblast-like cells for dentin repair. Thus, CGRP/Shh signaling should be considered for new strategies that seek to manage pain or dentin regeneration in the molar.

In addition to the action potentials generated by the ongoing activation of single dorsal horn neurons in the anesthetized cat, we often recorded small negative field potentials with a fast-rising phase and a slow decay (dIFPs). These potentials could be separated in different classes, each with a specific and rather constant shape and amplitude. They were largest in spinal laminae III-V and gradually faded at deeper locations, without showing the polarity reversal displayed at these depths by the focal potentials produced by stimulation of muscle and cutaneous afferents. We propose that the dIFPs are postsynaptic field potentials generated by strongly coupled sets of dorsal horn neurons displaying a spatial orientation that generates closed field potentials in response to stimulation of high-threshold cutaneous and muscle afferents. These neuronal sets could form part of the spinal inhibitory circuitry that mediates presynaptic inhibition and Ib non-reciprocal postsynaptic inhibition and could be involved in the sensory-motor transformations activated by stimulation of high-threshold cutaneous afferents.

It has been suggested that tactile intensity perception can be explained by a linear function of spike rate weighted by afferent type. Other than relying on mathematical models, verifying this experimentally is difficult due to the frequency tuning of different afferent types and changes in population recruitment patterns with vibrotactile frequency. To overcome these complexities, we used pulsatile mechanical stimuli which activate the same afferent population regardless of the repetition rate (frequency), generating one action potential per pulse. We used trains of different frequencies (20-200 Hz) to investigate perceived intensity. Subjects' magnitude ratings increased with pulse rate up to ∼100 Hz and plateaued beyond this frequency. This was true regardless of pulse amplitude, from small pulses that exclusively activated Pacinian (PC) afferents, to pulses large enough to activate other afferents including slowly adapting. Electrical stimulation, which activates afferents indiscriminately, plateaued at a similar frequency, although not in all subjects. As the plateauing did not depend on indentation magnitude and hence on afferent weights, we propose that the contribution of spike count to intensity perception is weighted by a function of frequency. This may explain why fine textures evoking high frequency vibrations of a small magnitude do not feel disproportionally intense.

Urinary tract infections (UTIs) cause bladder hyperactivity and pelvic pain, but the underlying causes of these symptoms remain unknown. We investigated whether afferent sensitization contributes to the bladder overactivity and pain observed in mice suffering from experimentally induced bacterial cystitis. Inoculation of mouse bladders with the uropathogenic Escherichia coli strain UTI89 caused pelvic allodynia, increased voiding frequency, and prompted an acute inflammatory process marked by leukocytic infiltration and edema of the mucosa. Compared with controls, isolated bladder sensory neurons from UTI-treated mice exhibited a depolarized resting membrane potential, lower action potential threshold and rheobase, and increased firing in response to suprathreshold stimulation. To determine whether bacterial virulence factors can contribute to the sensitization of bladder afferents, neurons isolated from naïve mice were incubated with supernatants collected from bacterial cultures with or depleted of lipopolysaccharide (LPS). Supernatants containing LPS prompted the sensitization of bladder sensory neurons with both tetrodotoxin (TTX)-resistant and TTX-sensitive action potentials. However, bladder sensory neurons with TTX-sensitive action potentials were not affected by bacterial supernatants depleted of LPS. Unexpectedly, ultrapure LPS increased the excitability only of bladder sensory neurons with TTX-resistant action potentials, but the supplementation of supernatants depleted of LPS with ultrapure LPS resulted in the sensitization of both population of bladder sensory neurons. In summary, the results of our study indicate that multiple virulence factors released from UTI89 act on bladder sensory neurons to prompt their sensitization. These sensitized bladder sensory neurons mediate, at least in part, the bladder hyperactivity and pelvic pain seen in mice inoculated with UTI89. NEW & NOTEWORTHY Urinary tract infection (UTI) produced by uropathogenic Escherichia coli (UPEC) promotes sensitization of bladder afferent sensory neurons with tetrodotoxin-resistant and tetrodotoxin-sensitive action potentials. Lipopolysaccharide and other virulence factors produced by UPEC contribute to the sensitization of bladder afferents in UTI. In conclusion, sensitized afferents contribute to the voiding symptoms and pelvic pain present in mice bladder inoculated with UPEC.

Sensory information is conveyed by populations of neurons, and coding strategies cannot always be deduced when considering individual neurons. Moreover, information coding depends on the number of neurons available and on the composition of the population when multiple classes with different response properties are available. Here, we study population coding in human tactile afferents by employing a recently developed simulator of mechanoreceptor firing activity. First, we highlight the interplay of afferents within each class. We demonstrate that the optimal afferent density to convey maximal information depends on both the tactile feature under consideration and the afferent class. Second, we find that information is spread across different classes for all tactile features and that each class encodes both redundant and complementary information with respect to the other afferent classes. Specifically, combining information from multiple afferent classes improves information transmission and is often more efficient than increasing the density of afferents from the same class. Finally, we examine the importance of temporal and spatial contributions, respectively, to the joint spatiotemporal code. On average, destroying temporal information is more destructive than removing spatial information, but the importance of either depends on the stimulus feature analyzed. Overall, our results suggest that both optimal afferent innervation densities and the composition of the population depend in complex ways on the tactile features in question, potentially accounting for the variety in which tactile peripheral populations are assembled in different regions across the body.

The cation channel TRPM3 is activated by heat and the neurosteroid pregnenolone sulfate. TRPM3 is expressed on sensory neurons innervating the skin, where together with TRPV1 and TRPA1, it functions as one of three redundant sensors of acute heat. Moreover, functional upregulation of TRPM3 during inflammation contributes to heat hyperalgesia. The role of TRPM3 in sensory neurons innervating internal organs such as the bladder is currently unclear. Here, using retrograde labeling and single-molecule fluorescent RNA in situ hybridization, we demonstrate expression of mRNA encoding TRPM3 in a large subset of dorsal root ganglion (DRG) neurons innervating the mouse bladder, and confirm TRPM3 channel functionality in these neurons using Fura-2-based calcium imaging. After induction of cystitis by injection of cyclophosphamide, we observed a robust increase of the functional responses to agonists of TRPM3, TRPV1, and TRPA1 in bladder-innervating DRG neurons. Cystometry and voided spot analysis in control and cyclophosphamide-treated animals did not reveal differences between wild type and TRPM3-deficient mice, indicating that TRPM3 is not critical for normal voiding. We conclude that TRPM3 is functionally expressed in a large proportion of sensory bladder afferent, but its role in bladder sensation remains to be established. ispartof: International Journal Of Molecular Sciences vol:23 issue:1 ispartof: location:Switzerland status: published

Recently it was documented that fatiguing, high-intensity exercise resulted in a significant attenuation in maximal skeletal muscle mitochondrial respiratory capacity, potentially due to the intramuscular metabolic perturbation elicited by such intense exercise. With the utilization of intrathecal fentanyl to attenuate afferent feedback from group III/IV muscle afferents, permitting increased muscle activation and greater intramuscular metabolic disturbance, this study aimed to better elucidate the role of metabolic perturbation on mitochondrial respiratory function. Eight young, healthy males performed high-intensity cycle exercise in control (CTRL) and fentanyl-treated (FENT) conditions. Liquid chromatography-mass spectrometry and high-resolution respirometry were used to assess metabolites and mitochondrial respiratory function, respectively, pre- and postexercise in muscle biopsies from the vastus lateralis. Compared with CTRL, FENT yielded a significantly greater exercise-induced metabolic perturbation (PCr: −67% vs. −82%, Pi: 353% vs. 534%, pH: −0.22 vs. −0.31, lactate: 820% vs. 1,160%). Somewhat surprisingly, despite this greater metabolic perturbation in FENT compared with CTRL, with the only exception of respiratory control ratio (RCR) (−3% and −36%) for which the impact of FENT was significantly greater, the degree of attenuated mitochondrial respiratory capacity postexercise was not different between CTRL and FENT, respectively, as assessed by maximal respiratory flux through complex I (−15% and −33%), complex II (−36% and −23%), complex I + II (−31% and −20%), and state 3CI+CII control ratio (−24% and −39%). Although a basement effect cannot be ruled out, this failure of an augmented metabolic perturbation to extensively further attenuate mitochondrial function questions the direct role of high-intensity exercise-induced metabolite accumulation in this postexercise response.

The review surveys various aspects of the plasticity of nerve fibers, in particular the prolonged increase in their excitability evoked by polarization, focusing on a long-lasting increase in the excitability of myelinated afferent fibers traversing the dorsal columns of the spinal cord. We review the evidence that increased axonal excitability 1) follows epidurally applied direct current (DC) as well as relatively short (5 or 10 ms) current pulses and synaptically evoked intrinsic field potentials; 2) critically depends on the polarization of branching regions of afferent fibers at the sites where they bifurcate and give off axon collaterals entering the spinal gray matter in conjunction with actions of extrasynaptic GABAA membrane receptors; and 3) shares the feature of being activity-independent with the short-lasting effects of polarization of peripheral nerve fibers. A comparison between the polarization evoked sustained increase in the excitability of dorsal column fibers and spinal motoneurons (plateau potentials) indicates the possibility that they are mediated by partly similar membrane channels (including noninactivating type L Cav++ 1.3 but not Na+ channels) and partly different mechanisms. We finally consider under which conditions transspinally applied DC (tsDCS) might reproduce the effects of epidural polarization on dorsal column fibers and the possible advantages of increased excitability of afferent fibers for the rehabilitation of motor and sensory functions after spinal cord injuries. NEW & NOTEWORTHY This review supplements previous reviews of properties of nerve fibers by surveying recent experimental evidence for their long-term plasticity. It also extends recent descriptions of spinal effects of DC by reviewing effects of polarization of afferent nerve fibers within the dorsal columns, the mechanisms most likely underlying the long-lasting increase in their excitability and possible clinical implications.

BACKGROUND Axo-axonic contacts onto central terminals of primary afferents modulate sensory inputs to the spinal cord. These contacts produce primary afferent depolarization (PAD), which serves as a mechanism for presynaptic inhibition, and also produce dorsal root reflexes (DRRs), which may regulate the excitability of peripheral terminals and second order neurons. We aimed to identify changes in these responses as a consequence of peripheral inflammation. METHODS In vitro spinal cord recordings of spontaneous activities in dorsal and ventral roots were performed in control mice and following paw inflammation. We also used pharmacological assays to define the neurotransmitter systems implicated in such responses. RESULTS Paw inflammation increased the frequency and amplitude of spontaneous dorsal root depolarizations, the occurrence of DRRs and the amplitude of ventral roots depolarizations. PAD was classified in two different patterns based on their relation to ventral activity: time-locked and independent events. Both patterns increased in amplitude after paw inflammation, and independent events also increased in frequency. The circuits that were responsible for this activity implicated both glutamatergic and GABAergic transmission. Adrenergic modulation differentially affected both types of PAD, and this modulation changed after paw inflammation. CONCLUSIONS Our findings suggest the existence of independent spinal circuits at the origin of PAD and DRRs. Inflammation modulates these circuits differentially, unveiling varied mechanisms of spinal sensitization. This in vitro approach provides an isolated model for the study of the mechanisms of central sensitization and for the performance of pharmacological assays with the purpose of identifying and testing novel antinociceptive targets. SIGNIFICANCE Spinal circuits modulate activity of primary afferents acting on central terminals. Under in vitro conditions, dorsal roots show spontaneous activity in the form of depolarizations and action potentials. Our findings are consistent with the existence of several independent generator circuits. Experimental paw inflammation reduced mechanical withdrawal threshold and significantly increased the spontaneous activity of dorsal roots, which may be secondary to an enhanced output of spinal generators. This can be considered as a novel sign of central sensitization.

The advantage of locally applied anesthetics is that they are not associated with the many adverse effects, including addiction liability, of systemically administered analgesics. This therapeutic approach has two inherent pitfalls: specificity and a short duration of action. Here, we identified nociceptor endocytosis as a promising target for local, specific, and long-lasting treatment of inflammatory pain. We observed preferential expression of AP2α2, an α-subunit isoform of the AP2 complex, within CGRP+/IB4- nociceptors in rodents and in CGRP+ dorsal root ganglion neurons from a human donor. We utilized genetic and pharmacological approaches to inhibit nociceptor endocytosis demonstrating its role in the development and maintenance of acute and chronic inflammatory pain. One-time injection of an AP2 inhibitor peptide significantly reduced acute and chronic pain-like behaviors and provided prolonged analgesia. We evidenced sexually dimorphic recovery responses to this pharmacological approach highlighting the importance of sex differences in pain development and response to analgesics., The authors show the endocytotic adaptor subunit called AP2A2 is differentially expressed in CGRP+ nociceptors. Locally inhibiting nociceptor endocytosis with a lipidated AP2 inhibitor peptide reduces acute and chronic pain-like behaviour in mice and rats, indicating prolonged analgesia.

After vibration, Hoffmann reflex (H reflex) amplitude is depressed; however, the mechanisms underlying these phenomena remain unknown. This study investigated the influence of frequency and duration of vibration on the H reflex amplitude, heteronymous facilitation of the tendon jerk (T wave) mediated by group Ia afferents, and cervicomedullary motor evoked potential (CMEP) amplitude in 18 healthy human subjects. The H reflex of the flexor carpi radialis (FCR) was induced by median nerve stimulation at the elbow, and the conditioning FCR stimulation enhanced the T wave of the biceps brachii (BB). After vibration was applied to the FCR muscle belly, the amplitudes of the H reflex and heteronymous facilitation of the T wave were depressed; these influences persisted after the removal of vibration in all subjects. For the H reflex, there was no difference in the amount of depression among the frequencies of vibration used (57, 77, and 100 Hz). Higher frequencies of vibration were associated with longer recovery times of postvibration depression, and a longer duration of vibration was associated with a longer recovery time of the depression. Similar results were observed for heteronymous facilitation of the T wave, suggesting that the depression is caused by a decrease in postsynaptic potentials evoked by Ia afferents in spinal motoneurons; it was probably due to reduction in the number of Ia afferents recruited by the median nerve stimulation. Moreover, because the FCR CMEP amplitude was depressed after vibration, vibration should affect the responsiveness of spinal motoneurons. These mechanisms are considered to contribute to the H reflex depression after vibration.

Nociceptive markers in mice have been identified in two distinct peptidergic and nonpeptidergic neurons in the dorsal root ganglion (DRG) and distributed in different laminae of the dorsal horn of the spinal cord. Recently, however, a study in humans showed a significant overlapping in these two populations. In this study, we investigated the distribution of various nociceptive markers in the lumbar DRG and spinal cord of the dromedary camel. Immunohistochemical data showed a remarkable percentage of total neurons in the DRG expressed IB4 binding (54.5%), calcitonin gene-related peptide (CGRP; 49.5%), transient receptor potential vanilloid 1 (TRPV1; 48.2%), and nitric oxide synthase (NOS; 30.6%). The co-localization data showed that 89.6% and 74.0% of CGRP- and TRPV1-labeled neurons, respectively, were IB4 positive. In addition, 61.6% and 84.2% of TRPV1- and NOS-immunoreactive neurons, respectively, were also co-localized with CGRP. The distribution of IB4, CGRP, TRPV1, substance P, and NOS immunoreactivities in the spinal cord were observed in lamina I and outer lamina II (IIo). Quantitative data showed that 82.4% of IB4-positive nerve terminals in laminae I and IIo were co-localized with CGRP, and 86.0% of CGRP-labeled terminals were co-localized with IB4. Similarly, 85.1% of NOS-labeled nerve terminals were co-localized with CGRP. No neuropeptide Y (NPY) or cholecystokinin (CCK) immunoreactivities were detected in the DRG, and no co-localization between IB4, NPY, and CCK were observed in the spinal cord. Our results demonstrate marked convergence of nociceptive markers in the primary afferent neurons in camels, which is similar to humans rather than the mouse. The data also emphasizes the importance of interspecies differences when selecting ideal animal models for studying nociception and treating chronic pain.

Primary sensory neurons are a heterogeneous population of cells able to respond to both innocuous and noxious stimuli. Like most neurons they are highly compartmentalised, allowing them to detect, convey and transfer sensory information. These compartments include specialised sensory endings in the skin, the nodes of Ranvier in myelinated axons, the cell soma and their central terminals in the spinal cord. In this review, we will highlight the importance of these compartments to primary afferent function, describe how these structures are compromised following nerve damage and how this relates to neuropathic pain.

Primary afferent fibers express extrasynaptic GABAA and GABAB receptors in the axons and soma. However, whether these receptors are tonically activated by ambient GABA and the source of the neurotransmitter is presently unknown. Here, we show that GABA release from dorsal root ganglia (DRG) does not depend on extracellular calcium, but depends upon calcium released from intracellular stores, and is mediated by Best1 channels. Using a preparation consisting of the spinal nerve in continuity with the DRG and the dorsal root, we found that endogenous GABA tonically activates GABA receptors, depressing the excitability of the primary afferents. In addition, using HPLC we found that GABA is released in the DRG, and by immunofluorescence microscopy we show the presence of GABA, the Best1 channel, and some enzymes of the putrescine pathway of GABA biosynthesis, in glutamine synthase- and GFAP-positive satellite glial cells. Last, we found that the blockade of the Best1 channel activity reduced the excitability of primary afferents and prevented the activation of the GABA receptors. These results suggest that satellite glial cells may be the source of endogenous GABA released in the DRG via Best1 channels, which tonically activates extrasynaptic GABA receptors.

The perceived intensity of a vibrotactile stimulus is thought to depend on single-neuron firing rates (rate coding) and the number of active afferents (population coding). Unaddressed until now is whether the temporal relation of individual spikes also conveys information about tactile intensity. We used cutaneous electro-tactile stimulation to investigate how the temporal structure of a fixed number of spikes in a 1-s train influenced the perception of intensity. Four mean spike rates spanning the flutter and vibratory hum range (36 Hz, 60 Hz; 120 Hz, 180 Hz) were tested, with spikes grouped into a regular pattern, or bursts of 2-6 spikes spaced 3 ms apart. To link a putative neural code to perception, perceived intensity was assessed in 16 human participants (aged 20-45; 4 females) using the psychophysical paradigm of magnitude estimation. Compound sensory nerve action potentials were recorded to assess any stimulus variation in afferent recruitment. The temporal structuring of a fixed number of spikes into periodic bursts of multiple spikes altered perceived intensity as a function of burst spike count. The largest increase was seen at 36 Hz, where the bursts of six spikes were rated 2.1 times stronger than the regularly spaced spikes [95% confidence interval (CI): 1.9-2.3]. The true increase is likely larger as temporal structuring of spikes into bursts had some negative effect on afferent recruitment. We conclude that the perceived intensity can be modulated by changing temporal features of afferent discharge even when normalized for the number of recruited afferents.bNEWamp; NOTEWORTHY/bStructuring a fixed number of spikes into temporal burst patterns evoke gradations of perceived intensity with burst spike count, emphasizing the importance of spike timing in primary afferents for shaping perception. This forms the basis for new strategies in communicating a range of intensity information to users of neural interfaces by simply varying the timing of spikes in nonspecific primary afferents using fixed-charge electric pulses, without requiring alterations in stimulation current or mean pulse frequency.

Integrative functions of spinal interneurons are well recognized but the relative role of different interneuronal populations in this process continues to be investigated. It therefore appeared useful to review the principles of integration of afferent information by the interneurons analyzed so far as these principles should apply also to those remaining to be analyzed. Considering the results of both functional and morphological studies of spinal interneurons and of the morphology and immunochemistry of afferent fibers that provide input to them, the following five basic principles of processing of afferent information by them will be outlined; 1) afferent information of any origin is forwarded to several neuronal populations, 2) information from any sources of input is distributed unevenly, 3) input from several sources is integrated by individual neurons as well as by their populations, 4) specific combinations of input are integrated by different neuronal populations, and 5) afferent input to spinal interneurons is only one of the features distinguishing their functional populations. As the spinal neuronal organization and properties of neurons and afferent fibers in the so far investigated species (cat, rodents, and primates) have been found to resemble, future studies using molecular techniques in the mouse should allow the new data to integrate with those of the preceding studies and the principles outlined earlier as well as any new ones should apply also in humans.

Aims To map sensory and pelvic postganglionic neurons from three different regions of the female rat urethra. Methods The neuronal tracer True Blue (TB) was injected into the pre-pelvic, pelvic, and clitoral regions of the urethra from female Wistar rats. Seven days after TB injection, TB+ cells from the dorsal root ganglia (DRGs) and the major pelvic ganglion (MPG) were examined. The number and morphometry of TB+ cells were determined. Results TB+ cells were mainly distributed in lumbar 1 (L1), lumbar 2 (L2), lumbar 6 (L6), and sacral 1 (S1) DRGs, and in the MPG. The mean number of sensory neurons was 1200 ± 143. TB injection in pre-pelvic and pelvic urethra labeled neurons in L1, L2, L6, and S1 DRGs. TB injection in clitoral urethra labeled neurons in L6 and S1 DRGs. L6 DRG contained >50% of the total urethral TB+ neurons, and ~80% of the clitoral region. The mean value of the total number of MPG TB+ neurons was 1217 ± 72. DRG and MPG neurons projecting to the urethra presented a somatotopic distribution. Conclusions The results demonstrated that L6 DRG is the major supplier of afferent innervation to the urethra, and that the distal urethral region is exclusively innervated by lower lumbosacral DRGs. Considering that electrical stimulation of sensory pudendal nerve improves overactive bladder, and that most of the sensory neurons in the distal urethra are from L6 DRG, electrical stimulation of this ganglion may be an innovative and effective neuromodulation therapy for neurogenic urinary dysfunctions.

An exaggerated exercise pressor reflex (EPR) causes excessive sympathoexcitation and exercise intolerance during physical activity in the chronic heart failure (CHF) state. Muscle afferent sensitization contributes to the genesis of the exaggerated EPR in CHF. However, the cellular mechanisms underlying muscle afferent sensitization in CHF remain unclear. Considering that voltage-gated potassium (Kv) channels critically regulate afferent neuronal excitability, we examined the potential role of Kv channels in mediating the sensitized EPR in male rats with CHF. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting experiments demonstrate that both mRNA and protein expressions of multiple Kv channel isoforms (Kv1.4, Kv3.4, Kv4.2, and Kv4.3) were downregulated in lumbar dorsal root ganglions (DRGs) of CHF rats compared with sham rats. Immunofluorescence data demonstrate significant decreased Kv channel staining in both NF200-positive and IB4-positive lumbar DRG neurons in CHF rats compared with sham rats. Data from patch-clamp experiments demonstrate that the total Kv current, especially I(A), was dramatically decreased in medium-sized IB4-negative muscle afferent neurons (a subpopulation containing mostly Aδ neurons) from CHF rats compared with sham rats, indicating a potential functional loss of Kv channels in muscle afferent Aδ neurons. In in vivo experiments, adenoviral overexpression of Kv4.3 in lumbar DRGs for 1 wk attenuated the exaggerated EPR induced by muscle static contraction and the mechanoreflex by passive stretch without affecting the blunted cardiovascular response to hindlimb arterial injection of capsaicin in CHF rats. These data suggest that Kv channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in CHF. NEW & NOTEWORTHY The primary finding of this manuscript is that voltage-gated potassium (Kv) channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in chronic heart failure (CHF). We propose that manipulation of Kv channels in DRG neurons could be considered as a potential new approach to reduce the exaggerated sympathoexcitation and to improve exercise intolerance in CHF, which can ultimately facilitate an improved quality of life and reduce mortality.

International audience; The purpose of this study was to determine the contributions of feedforward and feedback processes on grip force regulation and object orientation during functional manipulation tasks. One patient with massive somatosensory loss resulting from large fibre sensory neuropathy, and ten control participants were recruited. Three experiments were conducted: 1) perturbation to static holding; 2) discrete vertical movement; and 3) functional grasp and place. The availability of visual feedback was also manipulated to assess the nature of compensatory mechanisms. Results from experiment 1 indicated that both the deafferented patient and controls used anticipatory grip force adjustments prior to self-induced perturbation to static holding. The patient exhibited increased grip response time, but the magnitude of grip force adjustments remained correlated with perturbation forces in the self-induced and external perturbation conditions. In experiment 2, the patient applied peak grip force substantially in advance of maximum load force. Unlike controls, the patient's ability to regulate object orientation was impaired without visual feedback. In experiment 3, the duration of unloading, transport and release phases were longer for the patient, with increased deviation of object orientation at phase transitions. These findings show that the deafferented patient uses distinct modes of anticipatory control according to task constraints, and that responses to perturbations are mediated by alternative afferent information. The loss of somatosensory feedback thus appears to impair control of object orientation, while variation in the temporal organization of functional tasks may reflect strategies to mitigate object instability associated with changes in movement dynamics.

We previously reported P2X3 purinoceptor (P2X3)-expressing vagal afferent nerve endings with large web-like structures in the subserosal tissue of the antral lesser curvature, suggesting that these nerve endings were one of the vagal mechanoreceptors. The present study investigated the morphological relationship between P2X3-immunoreactive nerve endings and serosal ganglia in the rat gastric antrum by immunohistochemistry of whole-mount preparations using confocal scanning laser microscopy. P2X3-immunoreactive basket-like subserosal nerve endings with new morphology were distributed laterally to the gastric sling muscles in the distal antrum of the lesser curvature. Parent axons ramified into numerous nerve fibers with pleomorphic flattened structures to form basket-like nerve endings, and the parent axons were originated from large net-like structures of vagal afferent nerve endings. Basket-like nerve endings wrapped around the whole serosal ganglia, which were characterized by neurofilament 200 kDa-immunoreactive neurons with or without neuronal nitric oxide synthase immunoreactivity and S100B-immunoreactive glial cells. Furthermore, basket-like nerve endings were localized in close apposition to dopamine beta-hydroxylase-immunoreactive sympathetic nerve fibers immunoreactive for vesicular nucleotide transporter. These results suggest that P2X3-immunoreactive basket-like nerve endings associated with serosal ganglia are the specialized ending structures of vagal subserosal mechanoreceptors in order to increase the sensitivity during antral peristalsis, and are activated by ATP from sympathetic nerve fibers and/or serosal ganglia for the regulation of mechanoreceptor function.

Piezo2 mechanotransduction channel is a crucial mediator of sensory neurons for sensing and transducing touch, vibration, and proprioception. We here characterized Piezo2 expression and cell specificity in rat peripheral sensory pathway using a validated Piezo2 antibody. Immunohistochemistry using this antibody revealed Piezo2 expression in pan primary sensory neurons of dorsal root ganglia in naive rats, which was actively transported along afferent axons to both central presynaptic terminals innervating the spinal dorsal horn (DH) and peripheral afferent terminals in the skin. Piezo2 immunoreactivity (IR) was also detected in the postsynaptic neurons of the DH and in the motor neurons of the ventral horn, but not in spinal glial fibrillary acidic protein-positive and Iba1-positive glia. Notably, Piezo2-IR was clearly identified in peripheral nonneuronal cells, including perineuronal glia, Schwann cells in the sciatic nerve and surrounding cutaneous afferent endings, as well as in skin epidermal Merkel cells and melanocytes. Immunoblots showed increased Piezo2 in dorsal root ganglia ipsilateral to plantar injection of complete Freund's adjuvant, and immunostaining revealed increased Piezo2-IR intensity in the DH ipsilateral to complete Freund's adjuvant injection. This elevation of DH Piezo2-IR was also evident in various neuropathic pain models and monosodium iodoacetate knee osteoarthritis pain model, compared with controls. We conclude that (1) the pan neuronal profile of Piezo2 expression suggests that Piezo2 may function extend beyond simply touch or proprioception mediated by large-sized low-threshold mechanosensitive primary sensory neurons; (2) Piezo2 may have functional roles involving sensory processing in the spinal cord, Schwann cells, and skin melanocytes; and (3) aberrant Piezo2 expression may contribute pain pathogenesis.

ASP7962 is a small molecule inhibitor for the nerve growth factor (NGF) receptor, tropomyosin-related kinase A (TrkA). NGF contributes to the survival of sensory and sympathetic neurons through TrkA receptor activation. Gross, microscopic, and quantitative effects to the nervous system were evaluated following oral ASP7962 administration to Sprague Dawley rats for 4 weeks and 13 weeks and after a recovery period. Histopathological findings included reversible neuronal atrophy but no neuronal death in the sympathetic ganglia (cervicothoracic ganglion, cranial mesenteric ganglion or superior [cranial] cervical ganglion). Stereological analysis showed reversible decreased ganglion volume and/or decreased neuron size in the superior (cranial) cervical ganglion in both the 4-week and the 13-week repeated dose studies. There were no test article related changes in the brain, dorsal root ganglia with spinal nerve roots or trigeminal ganglia and no functional deficits. ASP7962 did not cause any detectable dysfunction of the sympathetic and sensory nervous system in either study.

Anatomical tracing studies examining the vagal system can conflate details of sensory afferent and motor efferent neurons. Here, we used a serotype of adeno-associated virus that transports retrogradely and exhibits selective tropism for vagal afferents, to map their soma location and central termination sites within the nucleus of the solitary tract (NTS). We examined the vagal sensory afferents innervating the trachea, duodenum, stomach, or heart, and in some animals, from two organs concurrently. We observed no obvious somatotopy in the somata distribution within the nodose ganglion. The central termination patterns of afferents from different organs within the NTS overlap substantially. Convergence of vagal afferent inputs from different organs onto single NTS neurons is observed. Abdominal and thoracic afferents terminate throughout the NTS, including in the rostral NTS, where the 7th cranial nerve inputs are known to synapse. To address whether the axonal labeling produced by viral transduction is so widespread because it fills axons traveling to their targets, and not just terminal fields, we labeled pre and postsynaptic elements of vagal afferents in the NTS . Vagal afferents form multiple putative synapses as they course through the NTS, with each vagal afferent neuron distributing sensory signals to multiple second-order NTS neurons. We observe little selectivity between vagal afferents from different visceral targets and NTS neurons with common neurochemical phenotypes, with afferents from different organs making close appositions with the same NTS neuron. We conclude that specific viscerosensory information is distributed widely within the NTS and that the coding of this input is probably determined by the intrinsic properties and projections of the second-order neuron.

The main question addressed in this study was whether the refractoriness of nerve fibres can be modulated by their depolarisation and, if so, whether depolarisation of nerve fibres evokes a long-term decrease in the duration of the refractory period as well as the previously demonstrated increase in their excitability. This was investigated on nerve fibres within the dorsal columns, dorsal roots and peripheral nerves in deeply anaesthetised rats in vivo. The results revealed major differences depending on the sites of fibre stimulation and polarisation. Firstly, the relative refractory period was found to be shorter in epidurally stimulated dorsal column fibres than in fibres stimulated at other sites. Secondly, the minimal effective interstimulus intervals reflecting the absolute refractory period were likewise shorter for nerve fibres within the dorsal columns even though action potentials evoked by the second of a pair of stimuli were similarly delayed with respect to the preceding action potentials at all the stimulation sites. Thirdly, the minimal interstimulus intervals were reduced by epidurally applied cathodal direct current polarisation but not at other stimulation sites. Consequently, higher proportions of dorsal column fibres could be excited at higher frequencies, especially following their depolarisation, at interstimulus intervals as short as 0.5-0.7 ms. The results demonstrate that epidural depolarisation results in long-lasting effects not only on the excitability but also on the refractoriness of dorsal column fibres. They also provide further evidence for specific features of afferent fibres traversing the dorsal columns previously linked to properties of their branching regions.

Innocuous touch sensation is mediated by cutaneous low-threshold mechanoreceptors (LTMRs). Aβ slowly adapting type 1 (SA1) neurons constitute one LTMR subtype that forms synapse-like complexes with associated Merkel cells in the basal skin epidermis. Under healthy conditions, these complexes transduce indentation and pressure stimuli into Aβ SA1 LTMR action potentials that are transmitted to the central nervous system, thereby contributing to tactile sensation. However, it remains unknown whether this complex plays a role in the mechanical hypersensitivity caused by peripheral nerve injury. In this study, we characterized the distribution of Merkel cells and associated afferent neurons across four diverse domains of mouse hind paw skin, including a recently described patch of plantar hairy skin. We also showed that in the spared nerve injury (SNI) model of neuropathic pain, Merkel cells are lost from the denervated tibial nerve territory, but are relatively preserved in nearby hairy skin innervated by the spared sural nerve. Utilizing a genetic Merkel cell knockout mouse model, we subsequently examined the importance of intact Merkel cell-Aβ complexes to SNI-associated mechanical hypersensitivity in skin innervated by the spared neurons. We found that in the absence of Merkel cells, mechanical allodynia was partially reduced in male mice, but not female mice, under sural-sparing SNI conditions. Our results suggest that Merkel cell-Aβ afferent complexes partially contribute to mechanical allodynia produced by peripheral nerve injury, and that they do so in a sex dependent manner. SIGNIFICANCE STATEMENT Merkel discs or Merkel cell-Aβ afferent complexes are mechanosensory end organs in mammalian skin. Yet, it remains unknown whether Merkel cells or their associated sensory neurons play a role in the mechanical hypersensitivity caused by peripheral nerve injury. We found that male mice genetically lacking Merkel cell-Aβ afferent complexes exhibited a reduction in mechanical allodynia after nerve injury. Interestingly, this behavioral phenotype was not observed in mutant female mice. Our study will facilitate understanding of mechanisms underlying neuropathic pain.

Neuropathic pain is pain caused by damage to the somatosensory nervous system. Both degenerating injured nerves and neighboring sprouting nerves can contribute to neuropathic pain. However, the mesoscale changes in cutaneous nerve fibers over time after the loss of the parent nerve has not been investigated in detail. In this study, we followed the changes in nerve fibers longitudinally in the toe tips of mice that had undergone spared nerve injury (SNI). Nav1.8-tdTomato, Thy1-GFP and MrgD-GFP mice were used to observe the small and large cutaneous nerve fibers. We found that peripheral nerve plexuses degenerated within 3 days of nerve injury, and free nerve endings in the epidermis degenerated within 2 days. The timing of degeneration paralleled the initiation of mechanical hypersensitivity. We also found that some of the Nav1.8-positive nerve plexuses and free nerve endings in the fifth toe survived, and sprouting occurred mostly from 7 to 28 days. The timing of the sprouting of nerve fibers in the fifth toe paralleled the maintenance phase of mechanical hypersensitivity. Our results support the hypotheses that both injured and intact nerve fibers participate in neuropathic pain, and that, specifically, nerve degeneration is related to the initiation of evoked pain and nerve sprouting is related to the maintenance of evoked pain.

The projections from the claustrum to cortical areas within and adjacent to the superior parietal lobule were studied in 10 macaque monkeys, using retrograde tracers, computerized reconstructions, and quantitative methods. In contrast with the classical view that posterior parietal areas receive afferents primarily from the dorsal and posterior regions of the claustrum, we found that these areas receive more extensive projections, including substantial afferents from the anterior and ventral regions of the claustrum. Moreover, our findings uncover a previously unsuspected variability in the precise regions of the claustrum that originate the projections, according to the target areas. For example, areas dominated by somatosensory inputs for control of body movements tend to receive most afferents from the dorsal-posterior claustrum, whereas those which also receive significant visual inputs tend to receive more afferents from the ventral claustrum. In addition, different areas within these broadly defined groups differ in terms of quantitative emphasis in the origin of projections. Overall, these results argue against a simple model whereby adjacency in the cortex determines adjacency in the sectors of claustral origin of projections and indicate that subnetworks defined by commonality of function may be an important factor in defining claustrocortical topography.

Bladder afferents play a pivotal role in bladder function such as urine storage and micturition as well as conscious sensations such as urgency and pain. Endocannabinoids are ligands of cannabinoid 1 and 2 (CB1 and CB2) receptors but can influence the activity of a variety of G protein-coupled receptors as well as ligand-gated and voltage-gated channels. It is still not known which classes of bladder afferents are influenced by CB1 and CB2 receptor agonists. This study aimed to determine the role of CB2 receptors in two major classes of afferents in the guinea pig bladder: mucosal and muscular-mucosal. The mechanosensitivity of these two classes was determined by an ex vivo extracellular electrophysiological recording technique. A stable analog of endocannabinoid anandamide, methanandamide (mAEA), potentiated the mechanosensitivity of mucosal bladder afferents in response to stroking. In the presence of a transient receptor potential vanilloid 1 antagonist (capsazepine), the effect of mAEA switched from excitatory to inhibitory. A selective CB2 receptor agonist, 4-quinolone-3-carboxyamide (4Q3C), significantly inhibited the mechanosensitivity of mucosal bladder afferents to stroking. In the presence of a CB2 receptor antagonist, the inhibitory effect of 4Q3C was lost. mAEA and 4Q3C did not affect responses to stretch and/or mucosal stroking of muscular-mucosal afferents. Our findings revealed that agonists of CB2 receptors selectively inhibited the mechanosensitivity of capsaicin-sensitive mucosal bladder afferents but not muscular-mucosal afferents. This may have important implications for understanding of the role of endocannabinoids in modulating bladder function and sensation in health and diseases.

Key points Muscle spindle afferents are slowly adapting low threshold mechanoreceptors that report muscle length and movement information critical for motor control and proprioception. The rapidly adapting cation channel PIEZO2 has been identified as necessary for muscle spindle afferent stretch sensitivity, but the properties of this channel suggest additional molecular elements are necessary for mediating the complex slowly adapting response of muscle spindle afferents. We report that glutamate increases muscle spindle afferent static sensitivity in an ex vivo mouse muscle nerve preparation, while blocking glutamate packaging into vesicles by the sole vesicular glutamate transporter, VGLUT1, either pharmacologically or by transgenic knock out of one allele of VGLUT1 decreases muscle spindle afferent static but not dynamic sensitivity. Our results confirm that vesicle-released glutamate is an important contributor to maintained muscle spindle afferent excitability and may suggest a therapeutic target for normalizing muscle spindle afferent function. Abstract Muscle spindle afferents are slowly adapting low threshold mechanoreceptors which have both dynamic and static sensitivity to muscle stretch. The exact mechanism by which these neurons translate muscle movement into action potentials is not well understood, although the PIEZO2 mechanically sensitive cation channel is essential for stretch sensitivity. PIEZO2 is rapidly adapting, suggesting the requirement for additional molecular elements to maintain firing during stretch. Spindle afferent sensory endings contain glutamate-filled synaptic-like vesicles which are released in a stretch and calcium dependent manner. Previous work has shown that glutamate can increase and a phospholipase-D coupled metabotropic glutamate antagonist can abolish firing during static stretch. Here we test the hypothesis that vesicle-released glutamate is necessary for maintaining muscle spindle afferent excitability during static but not dynamic stretch. To test this hypothesis, we used a mouse muscle-nerve ex vivo preparation to measure identified muscle spindle afferent responses to stretch and vibration. In C57BL/6 adult mice, bath applied glutamate significantly increased the firing rate during the plateau phase of stretch, but not during the dynamic phase of stretch. Blocking the packaging of glutamate into vesicles by the sole vesicular glutamate transporter, VGLUT1, either with xanthurenic acid or by using a transgenic mouse with only one copy of the VGLUT1 gene (VGLUT1+/- ) decreased muscle spindle afferent firing during sustained stretch, but not during vibration. Our results suggest a model of mechanotransduction where calcium entering the PIEZO2 channel can cause the release of glutamate from synaptic-like vesicles which then helps to maintain afferent depolarization and firing. This article is protected by copyright. All rights reserved.

The blood pressure response to exercise is exaggerated in type 2 diabetes mellitus (T2DM). However, the underlying mechanisms remain unclear. It is hypothesized that one mechanism mediating the potentiated cardiovascular response in T2DM is the sensitization of chemically sensitive afferent neurons by activation of metaboreceptors. To test this hypothesis, we examined transient receptor potential cation channel subfamily V member 1 (TRPV1)-induced cardiovascular responses in vivo and muscle afferent discharge ex vivo in T2DM rats. Additionally, TRPV1 and protein kinase C (PKC) protein levels in dorsal root ganglia (DRG) subserving skeletal muscle were assessed. For 14-16 weeks, Sprague-Dawley rats were given either a normal diet (control) or high fat diet in combination with a low dose (35 and 25 mg/kg) of streptozotocin (T2DM). Administration of capsaicin, TRPV1 agonist, in hindlimb evoked significantly greater increases in mean arterial pressure and renal sympathetic neve activity in decerebrated T2DM than control. In a muscle-nerve preparation, the discharge to capsaicin exposure in group IV afferents isolated from T2DM was likewise significantly augmented at a magnitude that was proportional to glucose concentration. Moreover, the discharge to capsaicin was potentiated by acute exposure of group IV afferents to a high glucose environment. T2DM showed significantly increased phospholyrated-TRPV1 and −PKCα levels in DRG neurons as compared with control. These findings suggest that group IV muscle afferents are sensitized by PKC-induced TRPV1 overactivity in early-stage T2DM with hyperglycemia and, thereby, may contribute to the potentiated circulatory response to TRPV1 activation in the disease.