Effect of muscle length on the modulation of H reflex and inhibitory mechanisms of Ia afferent discharges during passive muscle lengthening.

The effectiveness of activated Ia afferents to discharge ᵯC-motoneurons is decreased during passive muscle lengthening compared with static and shortening muscle conditions. Evidence suggests that these regulations are explained by (1) greater post-activation depression induced by homosynaptic post-activation depression (HPAD) and (2) primary afferent depolarization (PAD). It remains uncertain whether muscle length impacts the muscle lengthening-related aspect of regulation of the effectiveness of activated Ia afferents to discharge ᵯC-motoneurons, HPAD, PAD and heteronymous Ia facilitation (HF). We conducted a study involving 15 healthy young individuals. We recorded conditioned or non-conditioned soleus Hoffmann (H) reflex with electromyography (EMG) to estimate the effectiveness of activated Ia afferents to discharge ᵯC-motoneurons, HPAD, PAD and HF during passive lengthening, shortening and static muscle conditions at short, intermediate and long lengths. Our results show that the decrease of effectiveness of activated Ia afferents to discharge ᵯC-motoneurons and increase of post-activation depression during passive muscle lengthening occur at all muscle lengths. For PAD and HF, we found that longer muscle length increases the magnitude of regulation related to muscle lengthening. To conclude, our findings support an inhibitory effect (resulting from increased post-activation depression) of muscle lengthening and longer muscle length on the effectiveness of activated Ia afferents to discharge α-motoneurons. The increase in post-activation depression associated with muscle lengthening can be attributed to the amplification of Ia afferents discharge.

Primary Afferent Depolarization and the Gate Control Theory of Pain: A Tutorial Simulation.

The gate control theory of pain postulates that the sensation of pain can be reduced or blocked by closing a "gate" at the earliest synaptic level in the spinal cord, where nociceptive (pain) afferents excite the ascending interneurons that transmit the signal to the brain. Furthermore, the gate can be induced to close by stimulating touch afferents with receptive fields in the same general area as the trauma that is generating the pain (the "rub it to make it better" effect). A considerable volume of research has substantiated the theory and shown that a key mechanism mediating the gate is pre-synaptic inhibition, and that this inhibition is generated by depolarizing IPSPs in the nociceptor central terminals (primary afferent depolarization; PAD). Both pre-synaptic inhibition and depolarizing IPSPs are topics that students often regard as matters of secondary importance (if they are aware of them at all), and yet they are crucial to a matter of primary importance to us all - pain control. This report describes some simple computer simulations that illustrate pre-synaptic inhibition and explore the importance of the depolarizing aspect of the IPSPs. These concepts are then built into a model of the gate control of pain itself. Finally, the simulations show how a small change in chloride homeostasis can generate the dorsal root reflex, in which nociceptor afferents generate antidromic spikes which may increase neurogenic inflammation and actually exacerbate pain. The hope is that the simulations will increase awareness and understanding of a topic that is important in both basic neuroscience and medical neurology.

Synchronous firing of dorsal horn neurons at the origin of dorsal root reflexes in naïve and paw-inflamed mice.

Spinal interneurons located in the dorsal horn induce primary afferent depolarization (PAD) controlling the excitability of the afferent's terminals. Following inflammation, PAD may reach firing threshold contributing to maintain inflammation and pain. Our aim was to study the collective behavior of dorsal horn neurons, its relation to backfiring of primary afferents and the effects of a peripheral inflammation in this system. Experiments were performed on slices of spinal cord obtained from naïve adult mice or mice that had suffered an inflammatory pretreatment. Simultaneous recordings from groups of dorsal horn neurons and primary afferents were obtained and machine-learning methodology was used to analyze effective connectivity between them. Dorsal horn recordings showed grouping of spontaneous action potentials from different neurons in "population bursts." These occurred at irregular intervals and were formed by action potentials from all classes of neurons recorded. Compared to naïve, population bursts from treated animals concentrated more action potentials, had a faster onset and a slower decay. Population bursts were disrupted by perfusion of picrotoxin and held a strong temporal correlation with backfiring of afferents. Effective connectivity analysis allowed pinpointing specific neurons holding pre- or post-synaptic relation to the afferents. Many of these neurons had an irregular fast bursting pattern of spontaneous firing. We conclude that population bursts contain action potentials from neurons presynaptic to the afferents which are likely to control their excitability. Peripheral inflammation may enhance synchrony in these neurons, increasing the chance of triggering action potentials in primary afferents and contributing toward central sensitization.

Non-linear multimodal integration in a distributed premotor network controls proprioceptive reflex gain in the insect leg.

Producing context-appropriate motor acts requires integrating multiple sensory modalities. Presynaptic inhibition of proprioceptive afferent neurons1-4 and afferents of different modalities targeting the same motor neurons (MNs)5-7 underlies some of this integration. However, in most systems, an interneuronal network is interposed between sensory afferents and MNs. How these networks contribute to this integration, particularly at single-neuron resolution, is little understood. Context-specific integration of load and movement sensory inputs occurs in the stick insect locomotory system,6,8-12 and both inputs feed into a network of premotor nonspiking interneurons (NSIs).8 We analyzed how load altered movement signal processing in the stick insect femur-tibia (FTi) joint control system by tracing the interaction of FTi movement13-15 (femoral chordotonal organ [fCO]) and load13,15,16 (tibial campaniform sensilla [CS]) signals through the NSI network to the slow extensor tibiae (SETi) MN, the extensor MN primarily active in non-walking animals.17-19 On the afferent level, load reduced movement signal gain by presynaptic inhibition. In the NSI network, graded responses to movement and load inputs summed nonlinearly, increasing the gain of NSIs opposing movement-induced reflexes and thus decreasing the SETi and extensor tibiae muscle movement reflex responses. Gain modulation was movement-parameter specific and required presynaptic inhibition. These data suggest that gain changes in distributed premotor networks, specifically the relative weighting of antagonistic pathways, could be a general mechanism by which multiple sensory modalities are integrated to generate context-appropriate motor activity.

The corticospinal tract primarily modulates sensory inputs in the mouse lumbar cord.

It is generally assumed that the main function of the corticospinal tract (CST) is to convey motor commands to bulbar or spinal motoneurons. Yet the CST has also been shown to modulate sensory signals at their entry point in the spinal cord through primary afferent depolarization (PAD). By sequentially investigating different routes of corticofugal pathways through electrophysiological recordings and an intersectional viral strategy, we here demonstrate that motor and sensory modulation commands in mice belong to segregated paths within the CST. Sensory modulation is executed exclusively by the CST via a population of lumbar interneurons located in the deep dorsal horn. In contrast, the cortex conveys the motor command via a relay in the upper spinal cord or supraspinal motor centers. At lumbar level, the main role of the CST is thus the modulation of sensory inputs, which is an essential component of the selective tuning of sensory feedback used to ensure well-coordinated and skilled movement.

Nociception induces a differential presynaptic modulation of the synaptic efficacy of nociceptive and proprioceptive joint afferents.

A previous study has indicated that during the state of central sensitization induced by the intradermic injection of capsaicin, there is a gradual facilitation of the dorsal horn neuronal responses produced by stimulation of the high-threshold articular afferents that is counteracted by a concurrent increase of descending inhibitory actions. Since these changes occurred without significantly affecting the responses produced by stimulation of the low-threshold articular afferents, it was suggested that the capsaicin-induced descending inhibition included a preferential presynaptic modulation of the synaptic efficacy of the slow conducting nociceptive joint afferents (Ramírez-Morales et al., Exp Brain Res 237:1629-1641, 2019). The present study was aimed to investigate more directly the contribution of presynaptic mechanisms in this descending control. We found that in the barbiturate anesthetized cat, stimulation of the high-threshold myelinated afferents in the posterior articular nerve (PAN) produces primary afferent hyperpolarization (PAH) in the slow conducting (25-35 m/s) and primary afferent depolarization (PAD) in the fast conducting (40-50 m/s) articular fibers. During the state of central sensitization induced by capsaicin, there is a supraspinally mediated shift of the autogenic PAH to PAD that takes place in the slow conducting fibers, basically without affecting the autogenic PAD generated in the fast conducting afferents. It is suggested that the change of presynaptic facilitation to presynaptic inhibition induced by capsaicin on the slow articular afferents is part of an homeostatic process aimed to keep the nociceptive-induced neuronal activity within manageable limits while preserving the proprioceptive information required for proper control of movement.

Nicotinic receptor modulation of primary afferent excitability with selective regulation of Aδ-mediated spinal actions.

Somatosensory input strength can be modulated by primary afferent depolarization (PAD) generated predominantly via presynaptic GABAA receptors on afferent terminals. We investigated whether ionotropic nicotinic acetylcholine receptors (nAChRs) also provide modulatory actions, focusing on myelinated afferent excitability in in vitro murine spinal cord nerve-attached models. Primary afferent stimulation-evoked synaptic transmission was recorded in the deep dorsal horn as extracellular field potentials (EFPs), whereas concurrently recorded dorsal root potentials (DRPs) were used as an indirect measure of PAD. Changes in afferent membrane excitability were simultaneously measured as direct current (DC)-shifts in membrane polarization recorded in dorsal roots or peripheral nerves. The broad nAChR antagonist d-tubocurarine (d-TC) selectively and strongly depressed Aδ-evoked synaptic EFPs (36% of control) coincident with similarly depressed A-fiber DRP (43% of control), whereas afferent electrical excitability remained unchanged. In comparison, acetylcholine (ACh) and the nAChR agonists, epibatidine and nicotine, reduced afferent excitability by generating coincident depolarizing DC-shifts in peripheral axons and intraspinally. Progressive depolarization corresponded temporally with the emergence of spontaneous axonal spiking and reductions in the DRP and all afferent-evoked synaptic actions (31%-37% of control). Loss of evoked response was long-lasting, independent of DC repolarization, and likely due to mechanisms initiated by spontaneous C-fiber activity. DC-shifts were blocked with d-TC but not GABAA receptor blockers and retained after tetrodotoxin block of voltage-gated Na+ channels. Notably, actions tested were comparable between three mouse strains, in rat, and when performed in different labs. Thus, nAChRs can regulate afferent excitability via two distinct mechanisms: by central Aδ-afferent actions, and by transient extrasynaptic axonal activation of high-threshold primary afferents.NEW & NOTEWORTHY Primary afferents express many nicotinic ACh receptor (nAChR) subtypes but whether activation is linked to presynaptic inhibition, facilitation, or more complex and selective activity modulation is unknown. Recordings of afferent-evoked responses in the lumbar spinal cord identified two nAChR-mediated modulatory actions: 1) selective control of Aδ afferent transmission and 2) robust changes in axonal excitability initiated via extrasynaptic shifts in DC polarization. This work broadens the diversity of presynaptic modulation of primary afferents by nAChRs.

The activation of D2 and D3 receptor subtypes inhibits pathways mediating primary afferent depolarization (PAD) in the mouse spinal cord.

Somatosensory information can be modulated at the spinal cord level by primary afferent depolarization (PAD), known to produce presynaptic inhibition (PSI) by decreasing neurotransmitter release through the activation of presynaptic ionotropic receptors. Descending monoaminergic systems also modulate somatosensory processing. We investigated the role of D1-like and D2-like receptors on pathways mediating PAD in the hemisected spinal cord of neonatal mice. We recorded low-threshold evoked dorsal root potentials (DRPs) and population monosynaptic responses as extracellular field potentials (EFPs). We used a paired-pulse conditioning-test protocol to assess homosynaptic and heterosynaptic depression of evoked EFPs to discriminate between dopaminergic effects on afferent synaptic efficacy and/or on pathways mediating PAD, respectively. DA (10 µM) depressed low-threshold evoked DRPs by 43 %, with no effect on EFPs. These depressant effects on DRPs were mimicked by the D2-like receptor agonist quinpirole (35 %). Moreover, by using selective antagonists at D2-like receptors (encompassing the D2, D3, and D4 subtypes), we found that the D2 and D3 receptor subtypes participate in the quinpirole depressant inhibitory effects of pathways mediating PAD.

Activation of α-adrenoceptors depresses synaptic transmission of myelinated afferents and inhibits pathways mediating primary afferent depolarization (PAD) in the in vitro mouse spinal cord.

Somatosensory afferent transmission strength is controlled by several presynaptic mechanisms that reduce transmitter release at the spinal cord level. We focused this investigation on the role of α-adrenoceptors in modulating sensory transmission in low-threshold myelinated afferents and in pathways mediating primary afferent depolarization (PAD) of neonatal mouse spinal cord. We hypothesized that the activation of α-adrenoceptors depresses low threshold-evoked synaptic transmission and inhibits pathways mediating PAD. Extracellular field potentials (EFPs) recorded in the deep dorsal horn assessed adrenergic modulation of population monosynaptic transmission, while dorsal root potentials (DRPs) recorded at root entry zone assessed adrenergic modulation of PAD. We found that noradrenaline (NA) and the α1-adrenoceptor agonists phenylephrine and cirazoline depressed synaptic transmission (by 15, 14 and 22%, respectively). DRPs were also depressed by NA, phenylephrine and cirazoline (by 62, 30, and 64%, respectively), and by the α2-adrenoceptor agonist clonidine, although to a lower extent (20%). We conclude that NA depresses monosynaptic transmission of myelinated afferents onto deep dorsal horn neurons via α1-adrenoceptors and inhibits interneuronal pathways mediating PAD through the activation of α1- and α2-adrenoceptors. The functional significance of these modulatory actions in shaping cutaneous and muscle sensory information during motor behaviors requires further study.

Primary afferent-driven presynaptic inhibition of C-fiber inputs to spinal lamina I neurons.

Presynaptic inhibition of primary afferent terminals is a powerful mechanism for controlling sensory information flow into the spinal cord. Lamina I is the major spinal nociceptive projecting area and monosynaptic input from C-fibers to this region represents a direct pathway for transmitting pain signals to supraspinal centers. Here we used an isolated spinal cord preparation to show that this pathway is under control of the afferent-driven GABAergic presynaptic inhibition. Presynaptic inhibition of C-fiber input to lamina I projection and local-circuit neurons is mediated by recruitment of Aß-, Aδ- and C-afferents. C-fiber-driven inhibition of C-fibers functions as a feedforward mechanism, by which the homotypic afferents control sensory information flow into the spinal cord and regulate degree of the primary nociceptive afferent activation needed to excite the second order neurons. The presynaptic inhibition of C-fiber input to lamina I neurons may be mediated by both synaptic and non-synaptic mechanisms, and its occurrence and extent are quite heterogeneous. This heterogeneity is likely to be reflective of involvement of lamina I neurons in diverse circuitries processing specific modalities of sensory information in the superficial dorsal horn. Thus, our results implicate both low- and high-threshold afferents in the modulation of C-fiber input into the spinal cord.

Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury.

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain. We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons. To this aim, rats with persistent hyperalgesia were randomly divided into four groups. Rats in the control group received no treatment, and the rat sciatic nerve was only exposed in the sham group. Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide, an inhibitor of NKCC1, based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group. In the experiment measuring thermal withdrawal latency, bumetanide (15 mg/kg) was intravenously administered. In the patch clamp experiment, bumetanide (10 µg/µL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour, or bumetanide (5 µg/µL) was intrathecally injected. The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats. We found that the thermal withdrawal latency of rats was significantly decreased on days 7, 14, and 21 after model establishment. After intravenous injection of bumetanide, the reduction in thermal retraction latency caused by model establishment was significantly inhibited. Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7, 14, and 21 after model establishment. No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment. The Cl- (chloride ion) fluorescent probe technique was used to evaluate the change of Cl- concentration in dorsal root ganglion neurons of chronic constriction injury model rats. We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl- fluorescent probe whose fluorescence intensity decreases as Cl- concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment. The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased, and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment. After bumetanide administration, the above indicators were significantly suppressed. These results confirm that CCI can induce abnormal overexpression of NKCC1, thereby increasing the Cl- concentration in dorsal root ganglion neurons; this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia. In addition, bumetanide can achieve analgesic effects. All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital, College of Medicine, Shihezi University, China on February 22, 2017 (approval No. A2017-169-01).

Extrasynaptic α5GABAA receptors on proprioceptive afferents produce a tonic depolarization that modulates sodium channel function in the rat spinal cord.

Activation of GABAA receptors on sensory axons produces a primary afferent depolarization (PAD) that modulates sensory transmission in the spinal cord. While axoaxonic synaptic contacts of GABAergic interneurons onto afferent terminals have been extensively studied, less is known about the function of extrasynaptic GABA receptors on afferents. Thus, we examined extrasynaptic α5GABAA receptors on low-threshold proprioceptive (group Ia) and cutaneous afferents. Afferents were impaled with intracellular electrodes and filled with neurobiotin in the sacrocaudal spinal cord of rats. Confocal microscopy was used to reconstruct the afferents and locate immunolabelled α5GABAA receptors. In all afferents α5GABAA receptors were found throughout the extensive central axon arbors. They were most densely located at branch points near sodium channel nodes, including in the dorsal horn. Unexpectedly, proprioceptive afferent terminals on motoneurons had a relative lack of α5GABAA receptors. When recording intracellularly from these afferents, blocking α5GABAA receptors (with L655708, gabazine, or bicuculline) hyperpolarized the afferents, as did blocking neuronal activity with tetrodotoxin, indicating a tonic GABA tone and tonic PAD. This tonic PAD was increased by repeatedly stimulating the dorsal root at low rates and remained elevated for many seconds after the stimulation. It is puzzling that tonic PAD arises from α5GABAA receptors located far from the afferent terminal where they can have relatively little effect on terminal presynaptic inhibition. However, consistent with the nodal location of α5GABAA receptors, we find tonic PAD helps produce sodium spikes that propagate antidromically out the dorsal roots, and we suggest that it may well be involved in assisting spike transmission in general. NEW & NOTEWORTHY GABAergic neurons are well known to form synaptic contacts on proprioceptive afferent terminals innervating motoneurons and to cause presynaptic inhibition. However, the particular GABA receptors involved are unknown. Here, we examined the distribution of extrasynaptic α5GABAA receptors on proprioceptive Ia afferents. Unexpectedly, these receptors were found preferentially near nodal sodium channels throughout the afferent and were largely absent from afferent terminals. These receptors produced a tonic afferent depolarization that modulated sodium spikes, consistent with their location.

Pre-Synaptic Inhibition of Afferent Feedback in the Macaque Spinal Cord Does Not Modulate with Cycles of Peripheral Oscillations Around 10 Hz.

Spinal interneurons are partially phase-locked to physiological tremor around 10 Hz. The phase of spinal interneuron activity is approximately opposite to descending drive to motoneurons, leading to partial phase cancellation and tremor reduction. Pre-synaptic inhibition of afferent feedback modulates during voluntary movements, but it is not known whether it tracks more rapid fluctuations in motor output such as during tremor. In this study, dorsal root potentials (DRPs) were recorded from the C8 and T1 roots in two macaque monkeys following intra-spinal micro-stimulation (random inter-stimulus interval 1.5-2.5 s, 30-100 µA), whilst the animals performed an index finger flexion task which elicited peripheral oscillations around 10 Hz. Forty one responses were identified with latency < 5 ms; these were narrow (mean width 0.59 ms), and likely resulted from antidromic activation of afferents following stimulation near terminals. Significant modulation during task performance occurred in 16/41 responses, reflecting terminal excitability changes generated by pre-synaptic inhibition (Wall's excitability test). Stimuli falling during large-amplitude 8-12 Hz oscillations in finger acceleration were extracted, and sub-averages of DRPs constructed for stimuli delivered at different oscillation phases. Although some apparent phase-dependent modulation was seen, this was not above the level expected by chance. We conclude that, although terminal excitability reflecting pre-synaptic inhibition of afferents modulates over the timescale of a voluntary movement, it does not follow more rapid changes in motor output. This suggests that pre-synaptic inhibition is not part of the spinal systems for tremor reduction described previously, and that it plays a role in overall-but not moment-by-moment-regulation of feedback gain.

Role of presynaptic glutamate receptors in pain transmission at the spinal cord level.

Nociceptive primary afferents release glutamate, activating postsynaptic glutamate receptors on spinal cord dorsal horn neurons. Glutamate receptors, both ionotropic and metabotropic, are also expressed on presynaptic terminals, where they regulate neurotransmitter release. During the last two decades, a wide number of studies have characterized the properties of presynaptic glutamatergic receptors, particularly those expressed on primary afferent fibers. This review describes the subunit composition, distribution and function of presynaptic glutamate ionotropic (AMPA, NMDA, kainate) and metabotropic receptors expressed in rodent spinal cord dorsal horn. The role of presynaptic receptors in modulating nociceptive information in experimental models of acute and chronic pain will be also discussed.

Expression of gamma-aminobutyric acid type A receptor α2 subunit in the dorsal root ganglion of rats with sciatic nerve injury.

The γ-aminobutyric acid neurotransmitter in the spinal cord dorsal horn plays an important role in pain modulation through primary afferent-mediated presynaptic inhibition. The weakening of γ-aminobutyric acid-mediated presynaptic inhibition may be an important cause of neuropathic pain. γ-aminobutyric acid-mediated presynaptic inhibition is related to the current strength of γ-aminobutyric acid A receptor activation. In view of this, the whole-cell patch-clamp technique was used here to record the change in muscimol activated current of dorsal root ganglion neurons in a chronic constriction injury model. Results found that damage in rat dorsal root ganglion neurons following application of muscimol caused concentration-dependent activation of current, and compared with the sham group, its current strength and γ-aminobutyric acid A receptor protein expression decreased. Immunofluorescence revealed that γ-aminobutyric acid type A receptor α2 subunit protein expression decreased and was most obvious at 12 and 15 days after modeling. Our experimental findings confirmed that the γ-aminobutyric acid type A receptor α2 subunit in the chronic constriction injury model rat dorsal root ganglion was downregulated, which may be one of the reasons for the reduction of injury in dorsal root ganglion neurons following muscimol-activated currents.