Disease-Associated Variants in GRIN1, GRIN2A and GRIN2B genes: Insights into NMDA Receptor Structure, Function, and Pathophysiology.

N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptors critical for synaptic transmission and plasticity, and for the development of neural circuits. Rare or de-novo variants in GRIN genes encoding NMDAR subunits have been associated with neurodevelopmental disorders characterized by intellectual disability, developmental delay, autism, schizophrenia, or epilepsy. In recent years, some disease-associated variants in GRIN genes have been characterized using recombinant receptors expressed in non-neuronal cells, and a few variants have also been studied in neuronal preparations or animal models. Here we review the current literature on the functional evaluation of human disease-associated variants in GRIN1, GRIN2A and GRIN2B genes at all levels of analysis. Focusing on the impact of different patient variants at the level of receptor function, we discuss effects on receptor agonist and co-agonist affinity, channel open probability, and receptor cell surface expression. We consider how such receptor-level functional information may be used to classify variants as gain-of-function or loss-of-function, and discuss the limitations of this classification at the synaptic, cellular, or system level. Together this work by many laboratories worldwide yields valuable insights into NMDAR structure and function, and represents significant progress in the effort to understand and treat GRIN disorders. Keywords: NMDA receptor , GRIN genes, Genetic variants, Electrophysiology, Synapse, Animal models.

Primary afferent depolarization evoked by a painful stimulus.

Pulses of intense radiant heat applied to the plantar pad of unanesthetized spinal cats produced negative dorsal root potentials, increased excitability of cutaneous A fibers, and marked activation of ipsilateral flexor motoneurons. The same effects were obtained during cold block of A fiber conduction in the appropriate peripheral nerve. We conclude that adequate noxious activation of cutaneous C fibers depolarizes cutaneous A fibers.

Open channel block of NMDA receptor responses evoked by tricyclic antidepressants.

The action of desipramine (DMI) and promazine on the response of mouse hippocampal neurons to the excitatory amino acid N-methyl-D-aspartic acid (NMDA) was investigated using whole-cell and single-channel recording. DMI at 20-50 microM was a potent, selective antagonist of responses to NMDA but not kainate or quisqualate. At -60 mV, the Kd for DMI block of responses to NMDA was 10 microM. The potency of DMI as an NMDA antagonist was highly voltage-dependent and behaved as though the Kd increased e-fold per 36 mV depolarization, reflecting an increase in the dissociation rate constant. Prior block of NMDA receptors with Mg2+ prevented binding of DMI, suggesting an action in the open channel. Single-channel analysis showed a decrease in the open time and burst length distributions, consistent with binding of DMI to open channels. We suggest that the action of DMI on NMDA receptor channels is similar to that of MK-801 and does not reflect binding to other domains, such as the regulatory sites for Zn2+ and glycine.

Modulation of excitatory synaptic transmission by drugs that reduce desensitization at AMPA/kainate receptors.

Desensitization at AMPA/kainate receptors has been proposed to contribute to the decay of excitatory synaptic currents. We examined the action of aniracetam, wheat germ agglutinin (WGA), and concanavalin A (Con A), drugs that act via separate mechanisms to reduce desensitization evoked by L-glutamate in rat hippocampal neurons. The decay of excitatory synaptic currents, and sucrose-evoked miniature excitatory postsynaptic currents (EPSCs) was slowed 2- to 3-fold by aniracetam. In contrast, WGA increased the EPSC decay time constant only 1.3-fold and Con A had no effect. Aniracetam increased the magnitude of stimulus-evoked EPSCs 1.9-fold; variance analysis suggests a postsynaptic mechanism of action. WGA and Con A reduced EPSC amplitude via a presynaptic mechanism. Aniracetam increased the burst length of L-glutamate-activated single-channel responses. Simulations suggest that aniracetam either slows entry into a desensitized state or decreases the closing rate constant for ion channel gating.

Temperature dependence of NR1/NR2B NMDA receptor channels.

N-methyl-D-aspartate (NMDA) receptors are highly expressed in the CNS, mediate the slow component of excitatory transmission and play key roles in synaptic plasticity and excitotoxicity. These ligand-gated ion channels are heteromultimers composed of NR1 and NR2 subunits activated by glycine and glutamate. In this study, patch-clamp recordings were used to study the temperature sensitivity of recombinant NR1/NR2B receptors expressed in human embryonic kidney (HEK) 293 cells. Rate constants were assessed by fitting a six-state kinetic scheme to time courses of transient macroscopic currents induced by glutamate at 21.9-46.5 degrees C. Arrhenius transformation of the rate constants characterizing NMDA receptor channel activity indicates that the most sensitive were the rate constants of desensitization (temperature coefficient Q(10)=10.3), resensitization (Q(10)=4.6) and unbinding (Q(10)=3.6). Other rate constants and the amplitude of single-channel currents were less temperature sensitive. Deactivation of responses mediated by NR1/NR2B receptors after a brief application of glutamate was best fit by a double exponential function (tau(fast): Q(10)=3.7; tau(slow): Q(10)=2.7). From these data, we conclude that desensitization/resensitization of the NMDA receptor and glutamate unbinding are especially temperature sensitive and imply that at physiological temperatures the channel kinetics play an important role in determining amplitude and time course of NMDA receptor-mediated postsynaptic currents and these receptors mediated synaptic plasticity.

Calcium-dependent desensitization of vanilloid receptor TRPV1: a mechanism possibly involved in analgesia induced by topical application of capsaicin.

The rationale for the topical application of capsaicin and other vanilloids in the treatment of pain is that such compounds selectively excite and subsequently desensitize nociceptive neurons. This desensitization is triggered by the activation of vanilloid receptors (TRPV1), which leads to an elevation in intracellular free Ca2+ levels. Depending on the vanilloid concentration and duration of exposure, the Ca2+ influx via TRPV1 desensitizes the channels themselves, which may represent not only a feedback mechanism protecting the cell from toxic Ca2+ overload, but also likely contributes to the analgesic effects of capsaicin. This review summarizes the current state of knowledge concerning the mechanisms that underlie the acute capsaicin-induced Ca2+-dependent desensitization of TRPV1 channels and explores to what extent they may contribute to capsaicin-induced analgesia. In view of the polymodal nature of TRPV1, we illustrate how the channels behave in their desensitized state when activated by other stimuli such as noxious heat or depolarizing voltages. We also show that the desensitized channel can be strongly reactivated by capsaicin at concentrations higher than those previously used to desensitize it. We provide a possible explanation for a high incidence of adverse effects of topical capsaicin and point to a need for more accurate clinical criteria for employing it as a reliable remedy.

Neurosteroid modulation of ionotropic glutamate receptors and excitatory synaptic transmission.

Ionotropic glutamate receptors function can be affected by neurosteroids, both positively and negatively. N-methyl-D-aspartate (NMDA) receptor responses to exogenously applied glutamate are potentiated or inhibited (depending on the receptor subunit composition) by pregnenolone sulphate (PS) and inhibited by pregnanolone sulphate (3alpha5betaS). While PS effect is most pronounced when its application precedes that of glutamate, 3alpha5betaS only binds to receptors already activated. Synaptically activated NMDA receptors are inhibited by 3alpha5betaS, though to a lesser extent than those tonically activated by exogenous glutamate. PS, on the other hand, shows virtually no effect on any of the models of synaptically activated NMDA receptors. The site of neurosteroid action at the receptor molecule has not yet been identified, however, the experiments indicate that there are at least two distinct extracellularly located binding sites for PS mediating its potentiating and inhibitory effects respectively. Experiments with chimeric receptors revealed the importance of the extracellular loop connecting the third and the fourth transmembrane domain of the receptor NR2 subunit for the neurosteroid action. alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors are inhibited by both PS and 3alpha5betaS. These neurosteroids also affect AMPA receptors-mediated synaptic transmission, however, in a rather indirect way, through presynaptically located targets of action.

Morphology and physiology of lamina I neurons of the caudal part of the trigeminal nucleus.

It has been suggested that in mammals, trigeminal lamina I neurons play a role in the processing and transmission of sensory information from the orofacial region. We investigated the physiological and morphological properties of trigeminal subnucleus caudalis (Sp5C) lamina I neurons in slices prepared from the medulla oblongata of 13- to 15-day-old postnatal rats using patch-clamp recordings and subsequent biocytin-streptavidin-Alexa labeling. Twenty-five neurons were recorded and immunohistochemically stained. The Sp5C lamina I consisted of several types of neurons which, on the basis of their responses to somatic current injection, can be classified into four groups: tonic neurons, which fired throughout the depolarizing pulse; phasic neurons, which expressed an initial burst of action potentials; delayed onset neurons, which showed a significant delay of the first action potential; and single spike neurons, characterized by only one to five action potentials at the very beginning of the depolarizing pulse even at high levels of stimulation intensity. Electrical stimulation of the spinal trigeminal tract evoked AMPA receptor-mediated excitatory postsynaptic currents (EPSC) exhibiting a strong polysynaptic component. AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSC) were characterized by a 10-90% rise time of 0.50+/-0.06 ms and a decay time constant of 2.5+/-0.5 ms. The kinetic properties of NMDA receptor-mediated EPSCs were measured at +40 mV. The 10-90% rise time was 8+/-2 ms and the deactivation time constants were 94+/-31 and 339+/-72 ms, respectively. Intracellular staining and morphological analysis revealed three groups of neurons: fusiform, pyramidal, and multipolar. Statistical analysis indicated that the electrophysiological properties and morphological characteristics are correlated. Tonic and phasic neurons were fusiform or pyramidal and delayed onset and single spike neurons were multipolar. Our results show that both the physiological and morphological properties of Sp5C lamina I neurons exhibit significant differences, indicating their specific integration in the processing and transmission of sensory information from the orofacial region.

Functional changes in the vanilloid receptor subtype 1 channel during and after acute desensitization.

Agonist-induced desensitization of the transient receptor potential vanilloid receptor-1 (TRPV1) is one of the key strategies that offer a way to alleviate neuropathic and inflammatory pain. This process is initiated by TRPV1 receptor activation and the subsequent entry of extracellular Ca(2+) through the channel into sensory neurones. One of the prominent mechanisms responsible for TRPV1 desensitization is dephosphorylation of the TRPV1 protein by the Ca(2+)/calmodulin-dependent enzyme, phosphatase 2B (calcineurin). Of several consensus phosphorylation sites identified so far, the most notable are two sites for Ca(2+)/calmodulin dependent kinase II (CaMKII) at which the dynamic equilibrium between the phosphorylated and dephosphorylated states presumably regulates agonist binding. We examined the mechanisms of acute Ca(2+)-dependent desensitization using whole-cell patch-clamp techniques in human embryonic kidney (HEK) 293T cells expressing the wild type or CaMKII phosphorylation site mutants of rat TRPV1. The nonphosphorylatable mutant S502A/T704I was capsaicin-insensitive but the S502A/T704A construct was fully functional, indicating a requirement for a specific residue at position 704. A point mutation at the nearby conserved residue R701 strongly affected the heat, capsaicin and pH-evoked currents. As this residue constitutes a stringent CaMKII consensus site but is also predicted to be involved in the interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), these data suggest that in addition to dephosphorylation, or as its consequence, a short C-terminal juxtamembrane segment adjacent to the transient receptor potential box composed of R701 and T704 might be involved in the decelerated gating kinetics of the desensitized TRPV1 channel.

Subtype-dependence of N-methyl-D-aspartate receptor modulation by pregnenolone sulfate.

N-methyl-D-aspartate receptors play a critical role in synaptogenesis, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits. The subunit composition determines the biophysical and pharmacological properties of the N-methyl-D-aspartate receptor channel complex. In this study, we report that responses mediated by recombinant rat N-methyl-D-aspartate receptors expressed in human embryonic kidney HEK293 cells are differentially affected by naturally occurring neurosteroid pregnenolone sulfate. We show that responses induced by 1mM glutamate in NR1-1a/NR2A and NR1-1a/NR2B receptors are potentiated five- to eight-fold more by pregnenolone sulfate than responses of NR1-1a/NR2C and NR1-1a/NR2D receptors with no differences in the concentration of pregnenolone sulfate that produced 50% potentiation. In addition to potentiation, pregnenolone sulfate also has an inhibitory effect at recombinant N-methyl-D-aspartate receptors, with values of the concentration of pregnenolone sulfate that produces 50% inhibition of NR1/NR2D=NR1/NR2C

Physiology and pathology of NMDA receptors.

Ionotropic glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype are highly expressed in the central nervous system and are involved in excitatory synaptic transmission and synaptic plasticity. Prolonged activation of NMDA receptors can lead to excitotoxicity, which is implicated in the pathogenesis of neurodegeneration occurring in various acute and chronic disorders of the central nervous system. Recent advances in understanding the function, pharmacology, genetics and structure of NMDA receptors has promoted a search for new compounds that could be therapeutically used. These compounds act on agonist binding sites, either apart from them or directly within the ion channel pore. Members of the last group are called open channel blockers, and some of them, such as memantine and ketamine, are already clinically used. Kinetic modeling of NMDA receptor activity was employed to define the effects of various groups of modulators. Quantifying the action of these substances by kinetic parameters can help us to reveal the molecular mechanism of action at the receptor and to characterize the dependence of its action on the mode of NMDA receptor activation. Two modes are considered: phasic activation, induced by synaptically released glutamate, and tonic activation, which is expected to occur under pathological conditions when low, but sustained levels of glutamate activate NMDA receptors. The aim of our review is to summarize the recent data about the structural and functional properties of NMDA receptors and their role in long-term potentiation and excitotoxicity.

The effects of excessive heat on heat-activated membrane currents in cultured dorsal root ganglia neurons from neonatal rat.

The effects of high temperature (53-61 degrees C) on membrane currents (I(heat)) or depolarization (V(heat)) induced by noxious heat were studied in cultured dorsal root ganglia neurons from neonatal rats using the whole cell patch clamp technique. I(heat) or V(heat) produced by 3 s ramps of increasing temperature between 43 and 50 degrees C exhibited a fast slope (Q10>10) that was similar both during rising and falling temperature (n=85). Temperatures exceeding 52 degrees C resulted in slowdown in the recovery of I(heat), and the threshold for inducing I(heat) was shifted to lower temperatures in successive trials. These high temperatures (54-60 degrees C) caused a linear and incomplete recovery of I(heat) (Q10 decreased to <5; 4.5 +/- 0.4; n=17) and in successive trials the threshold of I(heat) decreased to temperatures close to that in the bath. The neurons, however, remained sensitive to capsaicin and to decreased extracellular pH. It is suggested that exposure of nociceptive neurons to excessive noxious heat results in an irreversible decrease of the energy barrier between the resting and activated state of the protein structures responsible for generation of I(heat). This may explain the sensitization of nociceptors after heat injury.

Oxidizing reagent copper-o-phenanthroline is an open channel blocker of the vanilloid receptor TRPV1.

The TRPV1 channel plays an important role in generating nociceptive signals in mammalian primary sensory neurons. It consists of 838 amino acids with six transmembrane segments (TM1-TM6), a pore-forming loop between TM5 and TM6 and N- and C- terminals located intracellularly. It is a homotetramer and forms a nonselective cationic channel that can be opened by capsaicin, weak acids and noxious heat. There are 18 cysteines (Cys), three of which are located on the extracellular side of the receptor in and around the region of the pore-forming loop. We report that the TRPV1 channel in transfected HEK293T cells and in cultured rat DRG neurons is blocked in the open state by an oxidizing agent Cu-o-phenanthroline complex (Cu:Phe). The effects of Cu:Phe are concentration dependent ( IC50 = 5.2 : 20.8 microm ) and fully reversible. Cu:Phe applied immediately before exposure to an acidic solution, capsaicin or noxious heat is without effect. Substitutions of the extracellular Cys residues (616, 621, 634) by glycine individually or together do not alter the blocking effects of Cu:Phe suggesting that disulfide cross-linking does not represent the underlying mechanism. It is suggested that the complex Cu:Phe, a bulky, positively charged molecule, represents a very effective and reversible open channel blocker of TRPV1.

Membrane currents induced by L-homocysteic acid in mouse cultured hippocampal neurons.

The concentration-response relationship of membrane currents induced by L-homocysteic acid was studied on mouse embryonic hippocampal neurons in culture (n = 56). In the majority of neurons two phases in the dose-response relationship could be distinguished. The first was characterized by responses to 3-100 microM L-homocysteic acid which desensitized with a time-constant greater than 1 s in a concentration-dependent manner and were antagonized by 30 microM D-L-2-amino-5-phosphonovaleric acid indicating activation of the N-methyl-D-aspartate receptors. At higher concentrations of L-homocysteic acid this component was strongly depressed. The second phase was characterized by sustained responses that were concentration-dependent (1 mM L-homocysteic acid maximum concentration tested) and were not blocked by D-L-2-amino-5-phosphonovaleric acid indicating activation of non-N-methyl-D-aspartate receptors. Eight neurons did not exhibit these two-phase characteristics in the concentration-response relationship at the beginning of the recording. The magnitude of responses to L-homocysteic acid was positively related to concentration and the responses were partially blocked by D-L-2-amino-5-phosphonovaleric acid. In these neurons, however, repeated applications of L-homocysteic acid at concentrations 30 microM up to 300 microM resulted in a long-lasting, three- to four-fold increase of the membrane current. This increase was completely blocked by D-L-2-amino-5-phosphonovaleric acid (50-100 microM) suggesting that it was produced by activation of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Axotomy-induced change in the properties of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor channels in rat motoneurons.

Properties of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor channels were studied in fluorescence-labelled control and axotomized motoneurons in spinal cord slices using a patch-clamp technique. Axotomy performed on the third postnatal day resulted in motoneuron death. Application of AMPA or kainate induced large whole-cell currents, but outside-out patches isolated from control motoneurons were either unresponsive or displayed only single-channel activity in response to rapid application of AMPA. Measurement of AMPA receptor channel openings in outside-out patches revealed multiple single-channel conductance levels: 12.2+/-1.0, 21. 9+/-1.5 and 32.6+/-3.2pS. In control motoneurons dialysed with spermine, the current-voltage relationship of responses induced by activation of AMPA receptor channels exhibited various degrees of inward rectification. The rectification index, the ratio of responses at +40 and -60mV, was used to compare the degree of inward rectification. The mean values of rectification index of responses to focal application of AMPA and AMPA receptor-mediated excitatory postsynaptic currents induced by focal electric stimulation were 0. 64+/-0.17 and 0.50+/-0.27, respectively. In axotomized motoneurons, the degree of rectification was significantly less for both responses induced by application of AMPA and for excitatory postsynaptic currents (0.91+/-0.09 and 0.95+/-0.12, respectively). Deactivation of AMPA receptors assessed from motoneuron excitatory postsynaptic currents at -70 mV was independent of postnatal age, with tau(fast)=0.88+/-0.35ms (A(fast)=78.2+/-11.8%) and tau(slow)=6. 3+/-3.2ms. In axotomized motoneurons, the decay time constants of excitatory postsynaptic currents were similar, tau(fast)=0.91+/-0. 42ms (A(fast)=85.8+/-12.6%) and tau(slow)=5.9+/-3.4ms. However, the mean amplitude of excitatory postsynaptic currents was only 43% of the amplitude recorded in control motoneurons. The results show that the current induced by activation of AMPA receptors in neonatal motoneurons is mediated by opening of both Ca(2+)-permeable and Ca(2+)-impermeable channels. As a result of axotomy, an experimental model of neurodegeneration, AMPA receptor channels in injured motoneurons destined to die become predominantly Ca(2+) impermeable. These findings suggest phenotypic control of AMPA receptor channel properties, presumably by affecting their subunit composition.

Reducing agent dithiothreitol facilitates activity of the capsaicin receptor VR-1.

The vanilloid receptor subtype 1 (VR1) is expressed in a sub-population of small dorsal root ganglion (DRG) neurones in mammals and serves as the common transducer of the pain-producing signals, such as noxious heat, acids and capsaicin [Caterina et al., Nature 389 (1997) 816-824; Tominaga et al., Neuron 21 (1998) 531-543]. On the extracellular side, VR1 has three cysteine residues at positions 616, 621 and 634. Here we report that dithiothreitol (DTT) (2-60 mM), an agent that maintains -SH groups of cysteines in a reduced state, greatly facilitates membrane currents induced by noxious heat or capsaicin (1 microM) in cultured DRG neurones from the rat and in VR1-transfected HEK293 cells. The effects of DTT are concentration-dependent and fully reversible. We suggest that the ratio between free sulfhydryl groups and disulfide bonds of the cysteine residues of VR1 pre-sets sensitivity of primary nociceptors to algogens and may represent a new target for treating some pain states in humans.

Intracellular spermine decreases open probability of N-methyl-D-aspartate receptor channels.

Spermine and related polyamines have been shown to be endogenous regulators of several ion channel types including ionotropic glutamate receptors. The effect of spermine on N-methyl-d-aspartate (NMDA) receptors in cultured rat hippocampal neurons was studied using single-channel and whole-cell patch clamp recordings. Intracellular spermine resulted in the dose-dependent inhibition of NMDA-induced responses. Spermine reversibly inhibited the single NMDA receptor channel activity in inside-out patches suggesting a membrane-delimited mechanism of action. Open probability of NMDA receptor channels was decreased in a dose-dependent manner. Mechanism of spermine-induced inhibition of NMDA receptor was different from that of intracellular Ca(2+)-induced NMDA receptor inactivation. Both pharmacological studies and single channel analysis indicate that in contrast to the effect of extracellular spermine the intracellular spermine effect is not dependent on the NMDA receptor subunit composition. We propose that intracellular spermine has a direct inhibitory effect on NMDA receptors that is different from calcium-induced NMDA receptor inactivation and spermine-induced voltage-dependent inhibition of AMPA/kainate receptors. Spermine-induced tonic change in the open probability of NMDA receptor channels may play a role in mechanisms underlying short-term changes in the synaptic efficacy.

A technique for fast application of heated solutions of different composition to cultured neurones.

A technique is described that allows the application of fast temperature changes (time constant approximately 300 ms) of solutions superfusing cultured neurones under whole-cell mode of membrane current recording. Its principle is in heating the common outlet of the manifold which consists of 12 tubes connected to barrels containing test solutions of different composition. The outlet is made from a glass capillary (25 mm length, 620/350 microns outer/inner diameter) coated on the outside wall with platinum for a length of 12 mm. The heating element, a platinum layer, is electrically connected to the probe fixed to the micromanipulator used for positioning the manifold. The solutions, driven by gravity, are applied by opening electronic valves controlled either manually or in programmed sequences. The DC current for heating is controlled either manually or by external voltage command. The advantage of the technique is that the same temperature pattern can be applied to 12 different solutions. The technique is used for classifying sensory neurones in culture with respect to their sensitivity to heat and algogens; however, it is applicable to any study of the effects of increased temperature on the activity of ion channels in cultured cells.

Evidence that excitatory amino acids not only activate the receptor channel complex but also lead to use-dependent block.

The effects of fast application of excitatory amino acids N-methyl-D-aspartate (NMDA), L-aspartate (ASP), L-glutamate (GLU), quisqualate (QU) and kainate (KAIN) were studied in neurons from the embryonic spinal cord of the chick in monolayer cultures by employing the 'patch clamp' technique in the 'whole cell' mode. It was found that NMDA, ASP, GLU and QU, but not KAIN, induced responses that exhibited several components. The early component decayed with a time constant of 2 s to a lower level of membrane current and discontinuation of the application was followed by an after-current which returned to the base-line with a time constant of about 7 s. It is suggested that NMDA, ASP, GLU and QU, but not KAIN, not only activate the receptor channel complex but also induce use-dependent block.

Specific and potassium components in the depolarization of the la afferents in the spinal cord of the cat.

In the cat spinal cord, primary afferent depolarization (PAD) of group Ia fibers of extensor muscles is produced by high-frequency stimulation (100 Hz) of group I muscle flexor afferents without significant increases in extracellular potassium. On the other hand, the PAD produced by stimulation of mixed and pure cutaneous nerves correlates well with increases in potassium ions. We conclude that the PAD produced by group I muscle afferents results from the activation of specific pathways making axo-axonic synapses with the Ia fiber terminals. The PAD of Ia fibers resulting from activation of cutaneous nerves involves instead unspecific accumulation of potassium ions.