Nerve Terminal Impulse Research Articles

Cyclosporine A Decreases Dryness-Induced Hyperexcitability of Corneal Cold-Sensitive Nerve Terminals.

Cyclosporine A (CsA) is used for the treatment of dry eye (DE) with good clinical results, improving tear secretion and decreasing subjective symptoms. These effects are attributed to the improved tear film dynamics, but there are no data on the effect of CsA on the abnormal sensory nerve activity characteristic in DE. Our purpose was to evaluate the CsA effect on the enhanced activity of corneal cold thermoreceptors in a tear-deficient DE animal model using in vitro extracellular recording of cold thermoreceptors nerve terminal impulses (NTIs) before and in the presence of CsA. NTI shape was also analyzed. Blinking frequency and tearing rate were also measured in awake animals before and after topical CsA. CsA increased the tearing and blinking of treated animals. CsA significantly decreased the peak response to cold of cold thermoreceptors. Neither their spontaneous NTIs discharge rate nor their cooling threshold were modified. CsA also seemed to reverse some of the changes in NTI shape induced by tear deficiency. These data suggest that, at least in part, the beneficial clinical effects of CsA in DE can be attributed to a direct effect on sensory nerve endings, although the precise mechanisms underlying this effect need further studies to be fully clarified.

Validation of the CD11c‐DTR transgenic mouse as a model to study neuroimmune interactions in the cornea

AbstractPurpose: CD11c‐DTR+ mice have a transgene encoding for a simian diphtheria toxin (DT) receptor under the CD11c promoter that makes dendritic cells (DCs) sensitive to DT, which causes DC depletion. Preliminary results using this mouse suggest that there is a functional interaction between DCs and corneal sensory nerves, as DC depletion modifies nerve activity and nociceptive behaviour. The aim of this work was to validate the CD11c‐DTR+ mouse model proving that the observed effects are not due to the DT itself, using littermates negative for the transgene and therefore resistant to DC depletion by DT.Methods: CD11c‐DTR+ and CD11c‐DTR‐ mice (5 months, both sexes) were used. Both groups received single or repetitive (every 2 days for 8 days) subconjunctival injections of DT or its vehicle (PBS). Basal tearing (in mm) was measured using phenol red threads placed in the temporally side of the eye for 30 s in isoflurane‐anaesthetised mice. To quantify spontaneous eye pain, the eye closure ratio was calculated by dividing the height by the width of the lid opening measured in photographs of freely moving animals. Nerve terminal impulse (NTI) activity of corneal cold thermoreceptor endings was recorded ex vivo in superfused eyes; spontaneous and stimulus‐evoked activity was explored.Results: Basal tearing was higher in DT‐injected CD11c‐DTR‐ animals, especially after repetitive injections (Day 0: 2 ± 0.16 mm vs. day 2: 2.8 ± 0.37 mm vs. day 8: 4.7 ± 0.36 mm, n = 5, p ≤ 0.001, RM ANOVA). Repetitive PBS injections caused similar effects in CD11c‐DTR+ and CD11c‐DTR‐ mice tearing (DTR−: 4.7 ± 0.36 mm, n = 5 vs. DTR+: 3.7 ± 1.48 mm, n = 3, p = 0.399, t‐test). No signs of spontaneous pain were observed in DT‐injected CD11c‐DTR‐ mice (Day 0: 0.91 ± 0.01 vs. 48 h: 0.93 ± 0.01 vs. day 8: 0.95 ± 0.01, n = 5, p = 0.065, RM ANOVA). No significant differences were found in spontaneous and stimulus‐evoked NTI activity recorded after repetitive injections of DT in CD11c‐DTR− mice or PBS in CD11c‐DTR+ mice.Conclusions: CD11c‐DTR+ mice are a valid model to study neuroimmune interactions. DT has no effects on pain behaviour or NTI activity of animals lacking the DT receptor. The changes in tearing appear to be consequence of the subconjunctival injection process per se.Reference PID2020‐115934RB‐I00 from DOI: MCIN/AEI/10.13039/50110001103

Piezo2 Mediates Low-Threshold Mechanically Evoked Pain in the Cornea.

Mammalian Piezo2 channels are essential for transduction of innocuous mechanical forces by proprioceptors and cutaneous touch receptors. In contrast, mechanical responses of somatosensory nociceptor neurons evoking pain, remain intact or are only partially reduced in Piezo2-deficient mice. In the eye cornea, comparatively low mechanical forces are detected by polymodal and pure mechanosensory trigeminal ganglion neurons. Their activation always evokes ocular discomfort or pain and protective reflexes, thus being a unique model to study mechanotransduction mechanisms in this particular class of nociceptive neurons. Cultured male and female mouse mechano- and polymodal nociceptor corneal neurons display rapidly, intermediately and slowly adapting mechanically activated currents. Immunostaining of the somas and peripheral axons of corneal neurons responding only to mechanical force (pure mechano-nociceptor) or also exhibiting TRPV1 (transient receptor potential cation channel subfamily V member 1) immunoreactivity (polymodal nociceptor) revealed that they express Piezo2. In sensory-specific Piezo2-deficient mice, the distribution of corneal neurons displaying the three types of mechanically evoked currents is similar to the wild type; however, the proportions of rapidly adapting neurons, and of intermediately and slowly adapting neurons were significantly reduced. Recordings of mechano- and polymodal-nociceptor nerve terminals in the corneal surface of Piezo2 conditional knock-out mice revealed a reduced number of mechano-sensitive terminals and lower frequency of nerve terminal impulse discharges under mechanical stimulation. Eye blinks evoked by von Frey filaments applied on the cornea were lower in Piezo2-deficient mice compared with wild type. Together, our results provide direct evidence that Piezo2 channels support mechanically activated currents of different kinetics in corneal trigeminal neurons and contributes to transduction of mechanical forces by corneal nociceptors.SIGNIFICANCE STATEMENT The cornea is a richly innervated and highly sensitive tissue. Low-threshold mechanical forces activate corneal receptors evoking discomfort or pain. To examine the contribution of Piezo2, a low-threshold mechanically activated channel, to acute ocular pain, we characterized the mechanosensitivity of corneal sensory neurons. By using Piezo2 conditional knock-out mice, we show that Piezo2 channels, present in the cell body and terminals of corneal neurons, are directly involved in acute corneal mechano-nociception. Inhibition of Piezo2 for systemic pain treatment is hindered because of its essential role for mechano-transduction processes in multiple body organs. Still, topical modulation of Piezo2 in the cornea may be useful to selectively relief unpleasant sensations and pain associated with mechanical irritation accompanying many ocular surface disorders.

Basal neuro‐immune interactions between resident dentritic cells and corneal sensory nerves

AbstractPurposeTo determine the influence of dendritic cells (DCs) in modulating corneal sensory nerve activity at basal conditions using a DC‐depletion mice model.MethodsC57BL/6 CD11c‐DTR mice (5‐6 month old) of both sexes were used. The effect of short‐term (2 days) and long‐term (8 days) DC depletion on corneal sensory nerve activity was studied in eyes following bilateral temporal and nasal subconjunctival injections of diphtheria toxin (DT) in anesthetized animals. Nerve terminal impulse (NTI) activity of corneal cold thermoreceptor endings was recorded in excised eyes continuously superfused with saline solution at 34ºC. Background and stimulus‐evoked (cooling and heating ramps) activity was studied. Naïve (no DT injections) CD11c‐DTR mice served as controls.ResultsShort‐term DC depletion produced a significant decrease in heating threshold (37.65±0.66 ºC, n=10; p=0.003, ANOVA) compared with naïve (41.57±1.77 ºC, n=4) and long‐term DC depletion conditions (42.27±0.97 ºC, n=8). However, long‐term DC depletion seemed to desensitize cold responses of sensory nerve endings, with higher cooling threshold and temperature to reach the peak frequency during the cooling ramp (30.65±0.48 ºC, n=17 vs. naïve, 32.31±0.29 ºC, n=20 and short‐term DC depletion, 32.2±0.44 ºC, n=17; p=0.005, ANOVA). Besides, in long‐term DC depleted eyes, NTI shape was altered: duration of NTIs was significantly increased (1.9± 0.06 ms, n=16; p=0.009, ANOVA) compared with naïve (1.5± 0.1 ms, n=18) and short‐term DC depleted eyes (1.7± 0.1 ms, n=11), having both longer depolarization and repolarization times.ConclusionsThe differences observed in both NTI firing and spike shape of cold nerve endings, suggest that depletion of resident DC alters corneal sensory nerve activity, therefore supporting the idea of a neuro‐immune interaction at basal conditions.(Support: SAF2017‐83674‐C2‐2‐R and ‐1‐R, AEI, Spain, and ERDF, EU; ACIF/2017/169 Fellowship from Generalitat Valenciana‐FSE to LR‐F)

Functional and Morphologic Alterations in Mechanical, Polymodal, and Cold Sensory Nerve Fibers of the Cornea Following Photorefractive Keratectomy.

To define the characteristics and time course of the morphologic and functional changes experienced by corneal sensory nerves after photorefractive keratectomy (PRK). Unilateral corneal excimer laser photoablation was performed in 54 anesthetized 3- to 6-month-old mice; 11 naïve animals served as control. Mice were killed 0, 3, 7, 15, and 30 days after PRK. Excised eyes were placed in a recording chamber superfused at 34°C. Electrical nerve impulse activity of single sensory terminals was recorded with a micropipette applied onto the corneal surface. Spontaneous and stimulus-evoked (cold, heat, mechanical, and chemical stimuli) nerve terminal impulse (NTI) activity was analyzed. Corneas were fixed and stained with anti-β-Tubulin III antibody to measure nerve density and number of epithelial nerve penetration points of regenerating subbasal leashes. Nerve fibers and NTI activity were absent in the injured area between 0 and 7 days after PRK, when sparse regenerating nerve sprouts appear. On day 15, subbasal nerve density reached half the control value and abnormally responding cold-sensitive terminals were recorded inside the lesion. Thirty days after PRK, nerve density was almost restored, active cold thermoreceptors were abundant, and polymodal nociceptor activity first reappeared. Morphologic regeneration of subbasal corneal nerves started shortly after PRK ablation and was substantially completed 30 days later. Functional recovery appears faster in cold terminals than polymodal terminals, possibly reflecting an incomplete damage of the more extensively branched cold-sensitive axon terminals. Evolution of postsurgical discomfort sensations quality may be associated with the variable regeneration pattern of each fiber type.

Inhibitory Effect of Amitriptyline on the Impulse Activity of Cold Thermoreceptor Terminals of Intact and Tear-Deficient Guinea Pig Corneas.

Chronic dryness of the ocular surface evokes sensitization of corneal cold-sensitive neurons through an increase of sodium currents and a decrease of potassium currents, leading to the unpleasant dryness and pain sensations typical of dry eye disease. Here, we explored the effects of amitriptyline, a voltage-gated Na+ channel blocker used for the treatment of depression and chronic pain, on nerve terminal impulse (NTI) activity of cold-sensitive nerve terminals recorded in intact and tear-deficient guinea pig corneas. Main lachrymal gland was surgically removed in anesthetized guinea pigs to induce chronic tear deficiency. Four to 6 weeks afterward, animals were sacrificed and both corneas placed in a perfusion chamber superfused at 34°C. Thermal stimuli were induced by changing the solution temperature from 34°C to 20°C (cooling ramp) and from 34°C to 50°C (heating ramp). Spontaneous and stimulus-evoked NTIs of cold-sensitive nerve terminals were recorded before, during, and after perfusion with solutions containing amitriptyline at different concentrations (3-30 μM). Perfusion with amitriptyline inhibited irreversibly and in a concentration-dependent manner the spontaneous NTI activity of cold thermoreceptors of intact corneas. This effect was less evident in tear-deficient corneas. In addition, amitriptyline (10 μM) attenuated the maximal response to cooling ramps without changing cold threshold in intact but not in tear-deficient corneas. Only cold thermoreceptors with low cooling threshold values were sensitive to amitriptyline. Amitriptyline effectively reduces the activity of cold thermoreceptors, although its efficacy is different in intact and tear-deficient corneas, which might be due to the changes induced by ocular dryness in the expression of the various voltage-gated Na+ channels responsible of the action potential generation and propagation.

Functional Properties of Sensory Nerve Terminals of the Mouse Cornea.

To define the firing properties of sensory nerve terminals innervating the adult mouse cornea in response to external stimuli of differing modality. Extracellular electrical activity of single corneal sensory nerve terminals was recorded in excised eyes of C57BL/6J mice. Eyes were placed in a recording chamber and were continuously superfused with warm saline solution. Nerve terminal impulse (NTI) activity was recorded by means of a glass pipette (tip ∼ 50 μm), applied on the corneal surface. Nerve terminal impulse discharges were stored in a computer for offline analysis. Three functionally distinct populations of nerve terminals were identified in the mouse cornea. Pure mechanonociceptor terminals (9.5%) responded phasically and only to mechanical stimuli. Polymodal nociceptor terminals (41.1%) were tonically activated by heat and hyperosmolal solutions (850 mOsm·kg-1), mechanical force, and/or TRPV1 and TRPA1 agonists (capsaicin and allyl isothiocyanate [AITC], respectively). Cold-sensitive terminals (49.4%) responded to cooling. Approximately two-thirds of them fired continuously at 34°C and responded vigorously to small temperature reductions, being classified as high-background activity, low-threshold (HB-LT) cold thermoreceptor terminals. The remaining one-third exhibited very low ongoing activity at 34°C and responded weakly to intense cooling, being named low-background activity, high-threshold (LB-HT) cold thermoreceptor terminals. The mouse cornea is innervated by trigeminal ganglion (TG) neurons that respond to the same stimulus modalities as corneal receptors of other mammalian species. Mechano- and polymodal endings underlie detection of mechanical and chemical noxious stimuli while HB-LT and LB-HT cold thermoreceptors appear to be responsible for basal and irritation-evoked tearing and blinking, respectively.

Effects of TRPM8 and TRPV1 agonists on the neural activity of corneal cold thermoreceptors in tear‐deficient guinea pigs

PurposeTo characterize the effects of menthol and capsaicin on the activity of corneal cold thermoreceptors in tear‐deficient guinea pigsMethodsThe main lachrymal gland was surgically removed in anesthetized animals. Four weeks later, cold‐sensitive nerve terminal impulse (NTI) activity was recorded from the superfused cornea. The spontaneous and stimulus‐evoked NTI activity was analysed. For thermal stimulation, temperature of perfusion solution was changed from 34°C (basal) down to 20°C (cooling ramp) or up to 50°C (heating ramp). TRP agonists (50–200 μM menthol, 1–10 μM capsaicin) were also added to the perfusion solution. The characteristics of the spontaneous and stimulus‐evoked NTI activity recorded in tear‐deficient and intact corneas before and during perfusion with TRP channel agonists were compared.ResultsRemoval of the lachrymal gland caused sustained reduction of basal and reflex tearing. NTI activity was significantly higher in cold thermoreceptors from tear‐deficient corneas compared with naïve corneas. Response to cooling ramps was also increased. Menthol increased ongoing and stimulus‐evoked activity of all cold thermoreceptors in both intact and tear deficient corneas. Menthol at high concentrations first excited and then inactivated cold thermoreceptors. Only 10% of thermoreceptors were activated by capsaicin.ConclusionsChronic tear deficiency alters the activity of corneal sensory nerve fibers, leading to the development of increased spontaneous activity and abnormal responsiveness to natural stimulation. These changes are particularly prominent in cold thermoreceptor fibers, whose injury‐evoked neuropathic firing seems to be due to altered expression of Na+ and K+ channels involved in impulse generation without changes in the activity of TRP channels involved in sensory transduction.

Lacosamide diminishes dryness-induced hyperexcitability of corneal cold sensitive nerve terminals

Lacosamide is an anti-epileptic drug that is also used for the treatment of painful diabetic neuropathy acting through voltage-gated sodium channels. The aim of this work was to evaluate the effects of acute application of lacosamide on the electrical activity of corneal cold nerve terminals in lacrimo-deficient guinea pigs.Four weeks after unilateral surgical removal of the main lachrimal gland in guinea pigs, corneas were excised and superfused in vitro at 34°C for extracellular electrophysiological recording of nerve terminal impulse activity of cold thermosensitive nerve terminals. The characteristics of the spontaneous and the stimulus-evoked (cooling ramps from 34°C to 15°C) activity before and in presence of lacosamide 100µM and lidocaine 100µM were compared.Cold nerve terminals (n=34) recorded from dry eye corneas showed significantly enhanced spontaneous activity (8.0±1.1 vs. 5.2±0.7imp/s; P<0.05) and cold response (21.2±1.7 vs. 16.8±1.3imp/s; P<0.05) as well as reduced cold threshold (1.5±0.1 vs. 2.8±0.2 Δ°C; P<0.05) to cooling ramps compared to terminals (n=58) from control animals. Both lacosamide and lidocaine decreased spontaneous activity and peak response to cooling ramps significantly (P<0.05). Temperature threshold was increased by the addition of lidocaine (P<0.05) but not lacosamide (P>0.05) to the irrigation fluid.In summary, the application of lacosamide results in a significant decrease of the augmented spontaneous activity and responsiveness to cold of corneal sensory nerves from tear-deficient animals. Based on these promising results we speculate that lacosamide might be used to reduce the hyperexcitability of corneal cold receptors caused by prolonged ocular surface dryness due to hyposecretory or evaporative dry eye disease.

Modulation of Smooth Muscle Relaxation by Short and Long Carbon Based Chemicals

Introduction: Short and long-chained based carbon chemicals derived from plants are found in many household products and are widely used in complementary medicine. It has been suggested anecdotally that some of these chemicals have the ability to relax smooth muscle tissues, and to test this concept, this study examines the effects of 1,8-cineole, 1-hepta-nol, α-pinene, cis-3-hexen-1-ol and trans-2-hexenal on neurotransmission and contraction of mouse and rat vas deferens. Methods: Focal extracel-lular recordings from the surface of mouse vas deferens of electrically-evoked nerve terminal impulses (NTIs) and electrically-evoked excitatory junctional currents (EJCs) were examined in both the presence and the absence of each chemical. Likewise, noradrenaline-evoked contractile forces of rat vas deferens were examined in both the presence and the absence of each chemical. Results: Relative to the respective controls, 1,8-cineole, 1-heptanol and trans-2-hexenal each statistically significantly decreased both smooth muscle contractile forces and EJC amplitudes, with 1-heptanol and trans-2-hexenal also statistically significantly decreasing NTI amplitudes. On NTI and EJC amplitudes the effects of 1,8-cineole and 1-heptanol were reversible whereas the effects of trans-2-hexenal were irreversible. Conclusion: Our results demonstrate that 1,8-cineole and trans-2-hexenal decrease smooth muscle neurotransmission and contraction and thereby cause smooth muscle relaxation, thus suggesting that these chemicals may have clinical applications and health benefits.

Effects of Lavender and Linalool on Neurotransmission and Contraction of Smooth Muscle

Introduction: Lavender (Lavandula angustifolia) is commonly used in household products, perfumes, aromatherapy and complementary medicines. This study assesses the effects of lavender and its component linalool on neurotransmission and contraction of smooth muscle. Methods: The concentration-dependent effects of lavender (0.001% to 0.05%) and linalool (0.001% to 0.05%) on electrically evoked nerve terminal impulse (NTI) and excitatory junction current (EJC) amplitudes were assessed, while the effects of lavender (0.03%) and linalool (0.03%) on 5-hydroxytryptamine, acetylcholine, histamine, noradrenaline and oxytocin evoked responses were examined. Reversibility of lavender (0.03%) and linalool (0.03%) effects on electrically evoked NTI and EJC amplitudes, as well as on acetylcholine evoked contractile responses, were also analysed. Results: Lavender and linalool caused concentration-dependent decreases of electrically evoked NTI and EJC amplitudes, and attenuated the contractile responses towards 5-hydroxytryptamine, acetylcholine, histamine, noradrenaline and oxytocin. Repeated washing of tissues treated with lavender following pre-treatment with acetylcholine reversed the inhibitory effects of lavender, whereas linalool’s effects were not readily reversible. Conclusion: Lavender and linalool may cause inhibition of smooth muscle presynaptic action potential propagation and postsynaptic G-protein coupled receptor evoked responses.

Tear fluid hyperosmolality increases nerve impulse activity of cold thermoreceptor endings of the cornea

Dry eye disease (DED) is a multifactorial disorder affecting the composition and volume of tears. DED causes ocular surface dryness, cooling, and hyperosmolality, leading ultimately to corneal epithelium damage and reduced visual performance. Ocular discomfort is the main clinical symptom in DED. However, the peripheral neural source of such unpleasant sensations is still unclear. We analyzed in excised, superfused mouse eyes, the effect of NaCl-induced hyperosmolality (325–1005mOsm·kg−1) on corneal cold thermoreceptor and polymodal nociceptor nerve terminal impulse (NTI) activity. Osmolality elevations at basal corneal temperature (33.6°C) linearly increased the ongoing NTI frequency of cold thermoreceptors, at a mean rate of 0.34imp·s−1/10mOsm. This frequency increase became significant with osmolality values greater than 340mOsm. Comparison of cold thermoreceptor activity increase induced by a dynamic temperature reduction of 1.8°C under iso- and hyperosmolal (360-mOsm) conditions provided evidence that more than 50% of the increased firing response was attributable to hyperosmolality. Comparatively, activation of corneal polymodal nociceptor endings by hyperosmolal solutions started with values of 600mOsm and greater. Sensitization of polymodal nociceptors by continuous perfusion with an “inflammatory soup” (bradykinin, histamine, prostaglandin E2 [PGE2], serotonin, and adenosine triphosphate [ATP]) did not enhance their activation by hyperosmolal solutions. High osmolality also altered the firing pattern and shape of cold and polymodal NTIs, possibly reflecting disturbances in local membrane currents. Results strongly suggest that tear osmolality elevations in the range observed in DED predominantly excite cold thermoreceptors, supporting the hypothesis that dryness sensations experienced by these patients are due, at least in part, to an augmented activity of corneal cold thermoreceptors.

Regeneration of functional nerves within full thickness collagen–phosphorylcholine corneal substitute implants in guinea pigs

Our objective was to evaluate promotion of tissue and nerve regeneration by extracellular matrix (ECM) mimics, using corneal implantation as a model system. Porcine type I collagen and 2-methacryloyloxyethyl phosphorylcholine (MPC) were crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and moulded into appropriate corneal dimensions to serve as substitutes for natural corneal ECM. These were implanted as full thickness grafts by penetrating keratoplasty into the corneas of guinea pigs after removal of the host tissue, and tracked over eight months, by clinical examination, slit-lamp biomicroscopy, and esthesiometry. Histopathology and ex vivo nerve terminal impulse recordings were performed at three months and at eight months. The implants promoted regeneration of corneal cells, nerves and the tear film, while retaining optical clarity. After three months, electrophysiological recordings showed evidence of mechano-nociceptors, and polymodal units inside the implants, while cold-sensitive units were present only on the peripheral host cornea. Following eight months, the incidence of nerve activity and the frequency of spontaneous firing were higher than in control eyes as reported for regenerating fibers. Active cold nerve terminals also innervated the implant area. We show that ECM mimetic materials can promote regeneration of corneal cells and functional nerves. The simplicity in fabrication and demonstrated functionality shows potential for ECM substitutes in future clinical applications.

Formula omitted] regenerative-loop potentiation of glutamate synaptic transmission by microglia and astrocytes

P 2 X 7 purinergic receptors have been implicated in chronic neuropathic and neuroinflammatory pain as well as in depression. These receptors are predominantly found in the central nervous system on microglial cells and on glutamatergic nerve terminals. Here, we develop hypotheses concerning mechanisms by which transient high-frequency impulse firing in glutamatergic terminals, such as occurs in nociceptor terminals accompanying neuropathic/neuroinflammatory pain, can lead to long-lasting changes in neural network function that is mediated by surrounding glial cells. The hypothesis consists of two parts. In the first, glutamate released by low-frequency (2 Hz) terminal action potentials is insufficient to generate postsynaptic action potentials, but these are generated by brief high-frequency input bursts. Glutamate released by these bursts is partly removed by transporters on the enveloping astrocyte processes and also excites AMPA receptors on these processes, which then release ATP. This ATP is partly metabolised to adenosine, which acts on presynaptic A1 receptors to inhibit glutamate release. The remaining ATP acts on the presynaptic P 2 X 7 receptors to facilitate glutamate release by both the high-frequency burst of action potentials as well as by a continuous low-frequency (2Hz) action potential firing that occurs in the absence of a neuropathic/neuroinflammatory insult. The positive feedback of terminal glutamate release, triggering astrocyte ATP release and leading to further glutamate release through activation of P 2 X 7 receptors, is then sufficient to allow the normal low-frequency (2 Hz) action potentials to now elicit postsynaptic action potentials after the insult is removed. In the second part of this model, the high concentration of ATP derived from astrocytes at the terminal attracts microglia by chemotaxis. The P 2 X 7 receptors on these microglia are then engaged, resulting in microglia secreting the cytokine TNF α . This acts on postsynaptic TNF-R1 receptors to increase the number of AMPA receptors there, thus enhancing the efficacy of synaptic transmission. The TNF α also acts on presynaptic TNF-R1 to increase the amount of glutamate released by each nerve terminal impulse. Experimental tests can be made of this hypothesis that P 2 X 7 receptors on the presynaptic terminal and those on the microglia synergistically act to ensure feedback pathways that reset to a high level the efficacy of synaptic transmission, thus ensuring chronic neuropathic/neuroinflammatory pain even when the initial insult has subsided.

Action potential initiation in the peripheral terminals of cold‐sensitive neurones innervating the guinea‐pig cornea

The site at which action potentials initiate within the terminal region of unmyelinated sensory axons has not been resolved. Combining recordings of nerve terminal impulses (NTIs) and collision analysis, the site of action potential initiation in guinea-pig corneal cold receptors was determined. For most receptors (77%), initiation mapped to a point in the time domain that was closer to the nerve terminal than to the site of electrical stimulation at the back of the eye. Guinea-pig corneal cold receptors are Adelta-neurones that lose their myelin sheath at the point where they enter the cornea, and therefore their axons conduct more slowly within the cornea. Allowing for this inhomogeneity in conduction speed, the resulting spatial estimates of action potential initiation sites correlated with changes in NTI shape predicted by simulation of action potentials initiating within a nerve terminal. In some receptors, more than one NTI shape was observed. Simulations of NTI shape suggest that the origin of differing NTI shapes result from action potentials initiating at different, spatially discrete, locations within the nerve terminal. Importantly, the relative incidence of NTI shapes resulting from action potential initiation close to the nerve termination increased during warming when nerve activity decreased, indicating that the favoured site of action potential initiation shifts toward the nerve terminal when it hyperpolarizes. This finding can be explained by a hyperpolarization-induced relief of Na(+) channel inactivation in the nerve terminal. The results provide direct evidence that the molecular entities responsible for stimulus transduction and action potential initiation reside in parallel with one another in the unmyelinated nerve terminals of cold receptors.

Barium ions inhibit the dynamic response of guinea-pig corneal cold receptors to heating but not to cooling.

An in vitro preparation of the guinea-pig cornea was used to study the effects of the K+ channel blockers 4-aminopyridine (4-AP), tetraethylammonium (TEA) and Ba2+ on nerve terminal impulses (NTIs) recorded extracellularly from cold sensory receptors. These receptors have an ongoing discharge of NTIs that is increased by cooling and decreased by heating. The K+ channel blocker 4-AP reduced the negative amplitude of the diphasic (positive-negative) NTIs, whereas TEA and Ba2+ prolonged the duration of the negative component. As the shape of the NTI is determined by the first derivative (dV/dt) of the membrane voltage change, these changes in the negative component are consistent with the blockade of K+ channels that contribute to action potential repolarization. Only TEA changed the basal activity of the receptors, increasing the likelihood of burst discharges. Ba2+ selectively reduced the response of the receptors to heating, whereas neither 4-AP nor TEA modified the response to heating or to cooling. The findings indicate that K+ channels blocked by 4-AP, TEA and Ba2+ contribute to action potential repolarization in corneal cold receptors, and that ionic mechanisms that underlie the reduction in NTI frequency in response to heating differ from those that increase activity in response to cooling.

Effects of Heating and Cooling on Nerve Terminal Impulses Recorded from Cold-sensitive Receptors in the Guinea-pig Cornea

An in vitro preparation of the guinea-pig cornea was used to study the effects of changing temperature on nerve terminal impulses recorded extracellularly from cold-sensitive receptors. At a stable holding temperature (31–32.5°C), cold receptors had an ongoing periodic discharge of nerve terminal impulses. This activity decreased or ceased with heating and increased with cooling. Reducing the rate of temperature change reduced the respective effects of heating and cooling on nerve terminal impulse frequency. In addition to changes in the frequency of activity, nerve terminal impulse shape also changed with heating and cooling. At the same ambient temperature, nerve terminal impulses were larger in amplitude and faster in time course during heating than those recorded during cooling. The magnitude of these effects of heating and cooling on nerve terminal impulse shape was reduced if the rate of temperature change was slowed. At 29, 31.5, and 35°C, a train of 50 electrical stimuli delivered to the ciliary nerves at 10–40 Hz produced a progressive increase in the amplitude of successive nerve terminal impulses evoked during the train. Therefore, it is unlikely that the reduction in nerve terminal impulse amplitude observed during cooling is due to the activity-dependent changes in the nerve terminal produced by the concomitant increase in impulse frequency. Instead, the differences in nerve terminal impulse shape observed at the same ambient temperature during heating and cooling may reflect changes in the membrane potential of the nerve terminal associated with thermal transduction.

The effects of polarizing current on nerve terminal impulses recorded from polymodal and cold receptors in the guinea-pig cornea.

It was reported recently that action potentials actively invade the sensory nerve terminals of corneal polymodal receptors, whereas corneal cold receptor nerve terminals are passively invaded (Brock, J.A., S. Pianova, and C. Belmonte. 2001. J. Physiol. 533:493–501). The present study investigated whether this functional difference between these two types of receptor was due to an absence of voltage-activated Na+ conductances in cold receptor nerve terminals. To address this question, the study examined the effects of polarizing current on the configuration of nerve terminal impulses recorded extracellularly from single polymodal and cold receptors in guinea-pig cornea isolated in vitro. Polarizing currents were applied through the recording electrode. In both receptor types, hyperpolarizing current (+ve) increased the negative amplitude of nerve terminal impulses. In contrast, depolarizing current (−ve) was without effect on polymodal receptor nerve terminal impulses but increased the positive amplitude of cold receptor nerve terminal impulses. The hyperpolarization-induced increase in the negative amplitude of nerve terminal impulses represents a net increase in inward current. In both types of receptor, this increase in inward current was reduced by local application of low Na+ solution and blocked by lidocaine (10 mM). In addition, tetrodotoxin (1 μM) slowed but did not reduce the hyperpolarization-induced increase in the negative amplitude of polymodal and cold nerve terminal impulses. The depolarization-induced increase in the positive amplitude of cold receptor nerve terminal impulses represents a net increase in outward current. This change was reduced both by lidocaine (10 mM) and the combined application of tetraethylammomium (20 mM) and 4-aminopyridine (1 mM). The interpretation is that both polymodal and cold receptor nerve terminals possess high densities of tetrodotoxin-resistant Na+ channels. This finding suggests that in cold receptors, under normal conditions, the Na+ conductances are rendered inactive because the nerve terminal region is relatively depolarized.

Electrophysiology of corneal cold receptor nerve terminals.

The mechanisms of sensory transduction in the fine nerve terminals of free nerve endings supplied by Adelta and C sensory axons are largely a matter of speculation. This is because the nerve terminals are small and inaccessible, particularly in intact tissues like skin. However, some of the difficulties associated with investigating the physiology of fine nerve terminals have recently been overcome using an in vitro preparation of the guinea-pig cornea that allows nerve terminal impulses (NTIs) to be recorded extracellularly from single polymodal and cold receptor nerve terminals. For cold receptors, the rate of spontaneously occurring NTIs is increased during cooling and decreased during heating. In addition, heating and cooling differentially modulate the shape of the recorded NTI. At the same temperature, NTIs are larger in amplitude and faster in time course during heating than those during cooling. The differential effect of heating and cooling on NTI shape is not considered to result simply from the temperature dependence of voltage-activated conductance kinetics or activity dependent changes in membrane excitability. Instead, changes in NTI shape may reflect changes in nerve terminal membrane potential that underlie the process of thermal transduction.

Effect of chronic clonidine treatment on transmitter release from sympathetic varicosities of the guinea-pig vas deferens.

1. Previous studies have demonstrated that chronic pre-synaptic inhibition of transmitter release by morphine evokes a counter-adaptive response in the sympathetic nerve terminals that manifests itself as an increase in transmitter release during acute withdrawal. In the present study we examined the possibility that other pre-synaptically acting drugs such as clonidine also evoke a counter-adaptive response in the sympathetic nerve terminals. 2. In chronically saline treated (CST) preparations, clonidine (0.5 microM) completely abolished evoked transmitter release from sympathetic varicosities bathed in an extracellular calcium concentration ([Ca(2+)](o)) of 2 mM. The inhibitory effect of clonidine was reduced by increasing [Ca(2+)](o) from 2 to 4 mM and the stimulation frequency from 0.1 to 1 Hz. 3. The nerve terminal impulse (NTI) was not affected by concentrations of clonidine that completely abolished evoked transmitter release. 4. Sympathetic varicosities developed a tolerance to clonidine (0.5 microM) following 7-9 days of chronic exposure to clonidine. 5. Acute withdrawal of preparations following chronic clonidine treatment (CCT) resulted in a significant (P < 0.005) enhancement of neurotransmitter release (3.75 times) above control levels observed in CST preparations. 6. The present findings demonstrate an enhancement of neurotransmitter release from sympathetic varicosities following acute withdrawal from chronic clonidine treatment.