Are unconventional NMDA receptors involved in slowly adapting type I mechanoreceptor responses?

Specific immunohistochemical staining for NMDA receptor NR2A/B subunits was found in the outer root sheath layer of rat sinus hair (whisker) follicle. Co-localization with CK 20 confirmed that Merkel cells were stained. The NR2A/B staining seen on Merkel cells was pericellular. In addition it appeared that NF70-positive staining was in close proximity to, but did not colocalise with NR2A/B immunoreactivity, indicating that NR2A/B was only expressed by Merkel cells and not their adjacent nerve terminals. Merkel cells and the nerve terminals have previously been associated with electrophysiological recordings from slowly adapting type I (St I) mechanoreceptor unit activity. Pharmacological experiments with isolated sinus hairs using a wide range of ionotropic glutamate receptor antagonists found that only certain NMDA receptor blockers depressed St I unit responses to mechanical stimuli. AMPA/kainate receptor antagonists (CNQX and NBQX, 100 microM) had no effect, nor did classical competitive NMDA receptor antagonists, D-AP5 (600 microM) and R-CPP (100 microM), nor the NMDA glycine site antagonist 5,7-dichlorokynurenic acid (100 microM). The only effective NMDA receptor blockers were those selective for the polyamine site: ifenprodil (IC50 20 microM) and Ro 25-6981 (IC50 approximately 50 microM), and the associated ion channel: MK 801, ketamine and (+/-)-1-(1,2-diphenylethyl)piperidine (IC50 < 100 microM). The two enantiomers of MK 801 were equipotent. All effects were long lasting, consistent with their non-/uncompetitive actions. The most potent drug tested, ifenprodil, at an effective dose of 30 microM, had a mean recovery time of 74 min. A three-fold increase in drug concentration was required to depress St II units (associated with non-synaptic lanceolate endings). Changes in Zn2+ did not affect St I unit responses. These data suggest that unconventional NMDA receptors are involved in St I unit responses, but question the notion of a glutamatergic synapse between the Merkel cell and nerve terminal.

[The Merkel cell: morphology, developmental origin, function]. / Merkelova bunka: morfologie, vývojový puvod, funkce.

Merkel nerve endings are mechanoreceptors in the vertebrate skin. They were named after the person who first described them--F.S. Merkel (1875). They consist of large, pale cells with lobulated nuclei forming synapse-like contacts with enlarged terminal endings of myelinated nerve fibres. Inside the cell are intermediate filaments formed of simple cytokeratins and osmophilic granules containing variety of neuropeptides. In mammals, they can be found in the basal layer of the skin and in those parts of the mucosa, which is derived from ectoderm. In contrast, in birds these cells are located in the dermis. The largest accumulation of Merkel nerve endings was found in whiskers of most mammals apart from man. There has been a controversy concerning the origin of Merkel cells. Results from chick/quail chimeras and most recently also from double transgenic mice have shown that Merkel cells are derived from the neural crest. Merkel cell play a role in the mechano-transduction process. In response to mechanical stimuli calcium ions enter Merkel cell and trigger the release of neurotransmitter probably glutamate. Thus, Merkel cell appears to be essential for characteristic slowly adapting response of these receptors during maintained mechanical stimuli. Cells in the skin of a similar appearance as Merkel cells are probably part of the diffuse neuroendocrine system and they do not function as mechanoreceptors. These cells, rather than those in mechanoreceptors, are most likely the origin of the highly malignant skin cancer called Merkel cell carcinoma.

Topography of muscle spindles and Golgi tendon organs in shoulder muscles of "Monodelphis domestica".

The topography of muscle spindles and Golgi tendon organs in the rotator cuff and surrounding shoulder muscles of a small laboratory marsupial (monodelphis domestica) were studied using light microscopy of serial sections. The shoulder joint of monodelphis has a large degree of freedom of movement allowing this animal to use the upper extremities for a wide range of activities like climbing and manipulating food. Thus, similar to the situation in man the shoulder joint is mainly secured by muscles. Silver stained serial paraffin sections were examined under the light microscope and the distribution of muscle spindles and Golgi tendon organs was reconstructed using three-dimensional image processing. In the two animals examined 113 and 131 muscle spindles respectively were found within the 4 rotator cuff muscles. In addition, 76 and 40 Golgi tendon organs respectively were seen at the musculo-tendinous junctions of these muscles preferentially close to the insertion at the humerus head. Also the surrounding shoulder muscles contain both muscle spindles and Golgi tendon organs in large numbers, but the ratio of Golgi tendon organs per muscle spindle appears to be lower. Number and localization of muscle spindles and Golgi tendon organs suggest, that these receptors are important for both reflex control of shoulder muscle tone as well as monitoring of static position and movement in the shoulder joint.

Topography of corpuscular mechanoreceptors in the shoulder joint region of monodelphis domestica.

The topography and structure of corpuscular mechanoreceptors in the shoulder joint capsule and periarticular connective tissue of a small laboratory marsupial (monodelphis domestica) were studied using light and electron microscopy. This animal is known to use its upper extremities for a wide range of activities like climbing and manipulating food. Thus, the shoulder joint of this animal species has a similar wide range of movement as the human shoulder joint, but is small enough for serial sectioning in its entirety. Silver stained serial paraffin sections were examined under the light microscope and the distribution of the different types of mechanoreceptors was reconstructed using three-dimensional image processing. In addition, selected mechanoreceptors were studied electron microscopically. Approximately 100 small lamellated corpuscles were found in the dense connective tissue of the joint capsule close to the insertion on the scapula and in the thickening of the joint capsule close to the glenoid labrum. Ruffini corpuscles were found in much smaller numbers in the moderately dense connective tissue of the axillary region. Only very few Vater-Pacinian corpuscles were seen in the soft periarticular connective tissue. The large number and localization of mechanoreceptor corpuscles in the shoulder joint capsule especially close to the glenoid labrum suggests, that these specialized nerve endings are likely to play an important role in control of joint movement. They can induce protective reflexes during extreme movements in the shoulder joint preventing shoulder luxation by increasing the tone of muscles pressing the humerus head into the glenoid cavity.

Sensory nerve endings in the hard palate and papilla incisiva of the goat.

The sensory innervation of the papilla incisiva in the hard palate of the domestic goat was studied with light and electron microscopy, supplemented by electrophysiological studies of free nerve endings. The goat lacks incisor teeth. Grass and leaves are not bitten, but pulled off by pressing them between the tongue and papilla incisiva. Thus, the masticatory mucosa is subject to particularly heavy mechanical loads requiring functional specialization of the horny epithelium in the form of thickening, i.e., the papilla incisiva and 12-14 pairs of rugae palatinae. A thin layer of firm connective tissue (lamina propria) attaches the mucosa to the periost of the hard palate. Sensory nerve fibers were found most abundantly in the papilla incisiva. Their number decreased drastically in aboral direction. A section through the first four rugae palatinae contains only about 10% of the number of free nerve endings found in the same area of mucosa from the papilla incisiva. Four types of sensory nerve endings were found. Free nerve endings were seen ubiquitously in the epithelium and superficial layer of the lamina propria. Merkel nerve endings were found in the bases of the epithelial thickenings in the papilla incisiva and rugae palatinae. Few Ruffini corpuscles were found in the deeper layer of the lamina propria, while lamellated corpuscles were seen just below the basement membrane of the epithelial pegs. Thus, a variety of sensory nerve endings were found in the hard palate, especially in those areas that are in close contact with the tongue during chewing of food. This rich innervation suggests an important role in monitoring the mechanical properties of food. Recordings were made from cell bodies supplying these terminals. Classic low-threshold, slowly adapting responses were observed in Ass afferent populations. This activity was probably mediated by Merkel type endings. Alternately, high-threshold and suprathreshold responses obtained from Adelta category afferents were likely to be nociceptive. In support of this, threshold and suprathreshold sensitization was observed following injection of serotonin into the receptive field of Adelta populations. This activity was likely to be derived from the aforementioned free nerve endings.

Distribution of sensory receptors in joints of the upper cervical column in the laboratory marsupial monodelphis domestica.

In order to investigate the sensory innervation, the upper cervical spine of a small laboratory marsupial (monodelphis domestica) was examined with serial section light microscopy and re-embedding of selected sections for electron microscopy. Large numbers of free nerve endings supplied by A delta- and C-fibres were found in the longitudinal ligaments and facet joint capsules. Electron microscopically, areas of direct contact between axon and collagen fibres of the surrounding connective tissue separated only by the basal lamina were observed. Such structural adaptations suggest mechanoreceptive or polymodal nociceptive functions. In addition, about 100 small lamellated corpuscles were found in the longitudinal ligaments mainly concentrated around the first intervertebral disk. Electron microscopy shows finger-like processes extending from the axon terminal into the inner core lamellae. These are the likely sites of the mechanoelectric transduction process. Smaller numbers of lamellated corpuscles were seen in the lower intervertebral disks and facet joint capsules. Lamellated corpuscles are known to function as rapidly adapting mechanoreceptors supplementing information supplied by muscle spindles to the CNS about position and movement of the cervical spine.

Sensory nerve endings in the hard palate and papilla incisiva of the rhesus monkey.

The sensory innervation of the hard palate of the rhesus monkey was studied by light and electron microscopy. The mucosa of the hard palate is subject to a particularly heavy mechanical load requiring functional specialisation of the horny epithelium in the form of thickenings - the papilla incisiva and eight pairs of rugae palatinae. A thin layer of firm connective tissue (lamina propria) attaches the mucosa to the periost of the hard palate. Sensory nerve fibres were found most abundantly in the papilla incisiva and first rugae palatinae. Their number decreases in an aboral direction. Five types of sensory nerve endings were found. Free nerve endings were ubiquitous in the epithelium and lamina propria. Merkel nerve endings were found in the basal layer of the epithelium of the papilla incisiva and rugae palatinae. Meissner corpuscles were located in the connective tissue between epithelial pegs, while lamellated corpuscles were seen below the epithelial pegs. Ruffini corpuscles were found in the deeper layer of the lamina propria. Thus, a variety of sensory nerve endings were found in the hard palate, especially in those areas that are in close contact with the tongue during chewing of food. This rich innervation suggests an important role in monitoring the mechanical properties of food and the position of the tongue.

Sensory nerve endings in the anterior cruciate ligament (Lig. cruciatum anterius) of sheep.

This study examines the structure of sensory nerve endings in the sheep anterior cruciate ligament (ACL). Three types of nerve endings are found: free nerve endings (FNE), Ruffini corpuscles, and lamellated corpuscles. The FNE (more than 100) are found subsynovially. The afferent nerve fibres are either thin myelinated axons (Adelta) or C fibres with diameters of 1-2 microm. FNE have been reported to function as thermoreceptors and polymodal nociceptors. In addition, FNE are also seen between fascicles of collagen fibres, often close to blood vessels. Part of this group may be efferent autonomic fibres controlling local blood flow. The corpuscles are seen subsynovially and between fascicles of connective tissue close to the attachment points of the ACL. A ligament contains about 20 Ruffini corpuscles, which are mainly located in the subsynovial connective tissue. They consist of cylinders formed from perineural cells surrounding the afferent myelinated axons (diameters 4-5 microm) with enlarged nerve terminals anchored between collagen fibres. These enter in bundles from the surrounding connective tissue at one open pole, pass through the length of the cylinder, and leave at the other pole. Functionally, Ruffini corpuscles have been described as slowly adapting stretch receptors. Lamellated corpuscles (usually between 5 and 15) are found in the subsynovial connective tissue. The afferent myelinated axon has a diameter of 4-6 microm, and the nerve terminal is located in the centre of numerous layers formed by lamellated terminal glial cells and by a perineural capsule. They are known to function as rapidly adapting pressure receptors. The most important function of the ACL is its mechanical function, but additional sensory functions must be considered triggering reflex mechanisms in case of extreme positioning or overload.

Functional evidence for calcium-induced calcium release in isolated rat vibrissal Merkel cell mechanoreceptors.

1. Single unit recordings were made from Merkel cell (sinus hair type I; St I) and sinus hair type II (St II) mechanoreceptors in isolated rat vibrissae. Responses were determined as the number of spikes evoked by controlled mechanical displacement of the hair shaft for 5 s every 30 s. 2. Superfusion of caffeine (10 mM) increased the responses of Merkel cell receptors by 50-180% of control (mean +/- S.E.M., 64 +/- 12.6%, n = 6, P < 0.001). Similar concentrations of caffeine inhibited St II receptor responses by 20-60% (mean +/- S.E.M., 35 +/- 8%, n = 5, P < 0.01). In both receptor types, caffeine induced a low-frequency increase in spontaneous firing. 3. When Merkel cell receptor responses were completely blocked by superfusion of high Mg2+-containing solution (to competitively block Ca2+ influx) caffeine had no effect when added after complete inhibition, but when added during partial inhibition of responses, the Mg2+-induced inhibition was transiently reversed or halted. This suggests that Ca2+ influx was a prerequisite for the action of caffeine. 4. Ryanodine (1 microM) increased the responses of Merkel cell receptors to mechanical stimulation by 7-60% (mean +/- S.E.M., 32 +/- 10.9 %, n = 5, P < 0.05) but had no effect on St II receptor responses. 5. The Ca2+-induced Ca2+ release (CICR) inhibitor procaine inhibited St I receptor responses in a concentration-dependent manner. Near-maximal inhibition was attained with 100 microM procaine. In four St I units, mean responses were depressed to 25% of control values. When both procaine (100 microM) and caffeine (10 mM) were introduced together, no net effect was seen. The responses of St II receptors were little affected by up to 100 microM procaine superfusion. 6. It is concluded that the mechano-electrical transduction process in St I receptors (but not St II) includes a CICR pathway. Taken with previous findings on the role of Merkel cells, it is likely that CICR is occurring in the Merkel cells.

Selective phototoxic destruction of quinacrine-loaded Merkel cells is neither selective nor complete.

Experiments were performed on slowly adapting type I mechanoreceptors in an isolated rat skin-nerve preparation (SA I receptors) and in an isolated rat sinus hair preparation (St I receptors). Merkel cells were stained in vitro with the fluorescent dye quinacrine and irradiated with ultraviolet (UV) light (2 mW for up to 1 h) while recording receptor responses to standard mechanical stimuli every 30 s. In addition, thresholds for electrically evoked action potentials were tested by applying electrical stimuli to the skin through the same stylus used for mechanical stimulation. UV irradiation resulted in abrupt failure to respond to mechanical stimuli in 73% of the SA I receptors examined (n = 37) within less than 1 h. This confirms previous reports of phototoxic destruction of Merkel cells. However, several minutes after the receptors failed to respond to mechanical stimulation, thresholds for electrical stimuli applied to the receptive field increased sharply. About 40% of the St I receptors (n = 13) irradiated with UV light following quinacrine staining stopped responding to bending of the hair within 1 h. In contrast, none of the seven St II receptors treated in the same way showed significant changes in the responses. Electron microscopic examination of sinus hairs after quinacrine staining alone showed slight changes in the appearance of Merkel cells, and in particular enlargement of the perinuclear space. These changes did not affect receptor responses. Electron microscopic studies of sinus hairs with receptors that had maintained normal responses to mechanical stimuli after quinacrine staining and 1 h of UV irradiation revealed that a substantial number of Merkel cells still had a normal ultrastructure while adjacent nerve terminals were severely swollen and partially compressing the Merkel cells. No changes were observed in lanceolate nerve terminals forming the morphological substrate of St II receptors. These results demonstrate that sensitivity to phototoxic destruction following quinacrine staining varies greatly among Merkel cells, with some maintaining normal function and ultrastructural appearance even after 1 h of UV irradiation. On the other hand there is clear evidence that the phototoxic damage affects the nerve terminals as well. Such experiments can therefore not provide conclusive proof about the role of Merkel cells in these mechanoreceptors.

Chloroquine specifically impairs Merkel cell mechanoreceptor function in isolated rat sinus hairs.

The function of Merkel cells in mechanotransduction has remained controversial Single unit recordings were made from Merkel cell receptors (sinus hair type I, St I) and another slowly adapting mechanoreceptor (sinus hair type II, St II) in isolated rat sinus hairs by applying controlled mechanical displacements to the hair shaft. Chloroquine (50-300 microM) caused a concentration dependent inhibition of Merkel cell receptor responses to mechanical stimulation. In contrast, both stimulated and spontaneous spike activity of St II receptors was increased by the same concentrations of chloroquine. Ultrastructural examination of chloroquine treated sinus hairs revealed swollen Merkel cells with multiple vacuoles and randomly distributed granules while other neural and surrounding structures showed no striking morphological changes. These results suggest that the Merkel cell plays a mechanotransducer role in Merkel cell receptors.

An isolated rat vibrissal preparation with stable responses of slowly adapting mechanoreceptors.

Sinus hairs were isolated from rats and examined in an isolated organ bath while superfused with oxygenated synthetic interstitial fluid. The distal end of the deep vibrissal nerve was teased for single unit recordings of responses from slowly adapting mechanoreceptors to standard bending of the hair. Sinus hair type I and type II receptors could be clearly identified by their respective characteristic firing pattern. Their responses were stable for at least 5 h even if the sinus hair had been stored at 4 degrees C for 24 h beforehand. Electron microscopic examination of these hairs at the end of experiments showed well preserved ultrastructure without abnormalities. The short diffusion distances in this preparation make it well suited for studying drug effects with the aim of investigating the mechanoelectric transduction process in these receptors.

Cytoplasmic Ca2+ concentrations in intact Merkel cells of an isolated, functioning rat sinus hair preparation.

An isolated, functioning sinus hair preparation was developed to investigate cytoplasmic Ca2+ concentrations in intact Merkel cells using microfluorimetric techniques. Intracellular Ca2+ levels were monitored by means of photon counters in small groups of Merkel cells loaded with the calcium fluorescent indicators fura-2 or fluo-3. Mechanical stimulation of Merkel cells with fine glass rods resulted in small transient increases in intracellular Ca2+ levels (by about 20%) in the group of Merkel cells around the stimulating probe. A rise in Ca2+ is presumed to be essential for the postulated synaptic transmission to the afferent nerve terminal. Depolarization with a high concentration of potassium chloride (100 mM) caused increases in intracellular Ca2+ concentrations in Merkel cells (by about 70%) only in the presence of extracellular Ca2+, indicating an influx of Ca2+ through voltage-gated channels. The Ca2+ response was abolished neither by (+)-BayK8644 nor omega-conotoxin, suggesting that the Ca2+ channels are different from the classical L- or N-type channels. Extracellular application of ATP (10 microM to 5 mM) caused dose-dependent increases in intracellular Ca2+ levels in Merkel cells of up to sevenfold from the basal level of about 100 nM. Similar responses to ATP were also measured during superfusion with Ca(2+)-free medium, suggesting intracellular stores as the main Ca2+ source. Pre-incubation of Merkel cells with the purinoceptor antagonist suramin (100 microM) for 30 min reduced the Ca2+ responses to ATP by about 50% compared with control conditions. In conclusion, the results have demonstrated that a rise in intracellular Ca2+ in Merkel cells can be evoked by mechanical stimulation, membrane depolarization and chemical stimulation by ATP. These observations strongly suggest a possible contribution of Ca2+ to the normal responsiveness of Merkel cell mechanoreceptors, in turn supporting the hypothesis that Merkel cells are involved in the mechano-electric transduction process in sinus hair type I mechanoreceptors.

Acute effects of neomycin on slowly adapting type I and type II cutaneous mechanoreceptors in the anaesthetized cat and rat.

1. Slowly adapting type I (SAI) and type II (SAII) mechanoreceptors in the skin were studied in anaesthetized cats and rats employing mechanical stimuli every 30 s. Individual stimuli rose within 200 ms to a plateau force which was kept constant through a feedback control unit for 2000 ms. 2. In cats, close arterial infusion of neomycin (2.5 mg/min) as sulphate was given through a side branch into the femoral blood stream for 5, 10 or 20 min at a rate of 0.025 ml/min. At other times saline was infused at the same rate. 3. After 20 min of neomycin infusion (total 50 mg) nervous discharge of cat SAI receptors was suppressed to about 30% of the control responses before neomycin infusion. Nervous responses were reduced more profoundly during the plateau phase of stimulation than during the dynamic phase. The interspike interval histogram was severely distorted. 4. In contrast, cat SAII receptors maintained about 70% of their control response after 20 min of neomycin infusion. The interspike interval histogram showed an orderly shift towards longer intervals maintaining its normal shape. 5. In rats, intradermal microinfusion of neomycin (30 micrograms/min) through a glass micropipette into the immediate vicinity of the receptor under investigation resulted in severe transient suppression of SAI receptor responses to about 10% of the control level. Receptor responses recovered almost completely about 1 h after the end of neomycin application. 6. It is concluded that the observed differences between the two types of slowly adapting mechanoreceptors are consistent with the hypothesis that the SAI receptor functions as a secondary sensory receptor, with a synaptic link between the Merkel cell and the primary afferent neurone.

Reduced responsiveness of touch (SA I) receptors in the cat following close arterial infusion of neomycin.

Electrophysiological recordings were made from afferent nerve fibres supplying slowly adapting type I (SA I) cutaneous mechano-receptors in the cat during application of mechanical stimuli (20 mN constant force, 0.2 s rise time and 2 s plateau phase, every 30 s). Close arterial infusion of neomycin (2.5 mg/min for 5 min) strongly depressed the responsiveness of SA I receptors. A direct effect on the receptor is thought to be the mode of action.

Mechanical properties of skin and responsiveness of slowly adapting type I mechanoreceptors in rats at different ages.

Slowly adapting type I (s.a. I) cutaneous mechanoreceptors were studied in young (3-4 months old) and adult (9-11 months old) rats. Trains of thirty repetitive mechanical stimuli with 0.1 s rise time, 1.9 s plateau phase, and 0.7 s interstimulus interval were applied. A feed-back mechanism maintained the force of stimulation at 20 mN during the plateau phases of stimuli and the contact force between stimuli at 0.5 mN. During the first few stimuli in a train residual indentation at contact force increased rapidly. Maximal indentation required to maintain the force of stimulation of 20 mN increased as well but to a smaller extent. Thus, the stroke amplitudes of individual stimuli decreased with increasing stimulus number. All displacement values in the group of adult rats were consistently reduced to 62 +/- 3% of the respective values in the group of young rats, indicating a linear decrease in skin compliance in the force range of 0.5-20 mN. Nervous responses to individual stimuli decreased from about 200 impulses for stimulus number 1 to about 60 impulses for stimulus number 30. Significant differences in the number of impulses between young and adult rats were observed from stimulus number 9 to number 16 only. It is concluded that the design of the s.a. I receptor allows maintained high tactile sensitivity in response to force-related stimuli irrespective of age-induced changes in mechanical properties of the skin and underlying tissues.

Responsiveness and ultrastructure of slowly adapting type I cutaneous mechanoreceptors in vitamin A deficient rats.

Single-unit recordings were made from afferent nerve fibres supplying slowly adapting type I (s.a. I) cutaneous mechanoreceptors in anaesthetized vitamin A deficient and control rats. Trains of thirty repetitive mechanical stimuli with 0.1 s rise time, 1.9 s plateau phase, and 0.7 s interstimulus interval were applied. A feed-back mechanism maintained the force of stimulation at 20 mN during the plateau phases and the contact force between stimuli at 0.5 mN. All displacement values in the group of vitamin A deficient rats were significantly larger than the corresponding control values. Residual indentations were increased by 70-100% while maximal indentations were only about 40% higher. These results indicate a non-linear increase in compliance of the skin and underlying tissues. S.a. I receptors were found to be significantly less responsive in vitamin A deficient animals. Mean numbers of impulses were about 25% lower in the vitamin A deficient group than in controls throughout the entire train of thirty stimuli. In vitamin A deficient rats, Merkel cells and adjoining nerve terminals showed signs of degeneration of a variety of cell organelles, particularly the mitochondria. Degenerative changes induced by vitamin A deficiency especially in the Merkel cells appeared to be a major cause of the reduction of responsiveness in s.a. I receptors.