Spinal termination of functionally identified primary afferent neurons with slowly conducting myelinated fibers.

Single primary afferent myelinated fibers from cutaneous receptors of cat and monkey were functionally identified by recording from the spinal cord with micropipettes filled with horseradish peroxidase (HRP). Relatively slowly conducting fibers (less than 40 m/sec) from high threshold mechanoreceptors (mechanical nociceptors) and two types of low threshold mechanoreceptor (D-hair and field) were selected for staining. Iontophoresis of the HRP and subsequent histochemical reaction stained the axons recorded from and their collaterals, including terminations, for several millimeters. The termination patterns in the two species proved essentially identical. Ipsilaterally, the mechanical nociceptor fibers terminated principally in the dorsal horn's marginal zone and in the ventral parts of the nucleus proprius (lamina V in the cat). Some of these nociceptors also had terminals in the midline just dorsal to the central canal, contralaterally in the marginal zone, and at the base of the opposite nucleus proprius. In contrast, the D-hair primary afferent axons terminated in the dorsal part of the nucleus proprius overlapping into the innermost portion of the substantia gelatinosa. The field receptor fibers terminated predominantly in the middle part of the nucleus proprius. These results suggest that there is a highly specialized central projection of primary afferent endings which is related to sensory function and not to fiber diameter. The marginal zone and most dorsal parts of the substantia gelatinosa receive direct projections from cutaneous nociceptors but do not have direct input from cutaneous receptors transmitting activity initiated by innocuous stimulation.     

Activation of fine articular afferent units by bradykinin

The responses of single fine articular afferent units to close intra-arterial injection of KCl and bradykinin were recorded from filaments of the saphenous nerve of the cat's right hindlimb. All units included in this study were sensitive to local mechanical probing of the medial and anteromedial aspects of the knee joint. The units were identified by conduction velocity as belonging either to group III (2.5-20 m/s, 17 units) or group IV (less than 2.5 m/s, 23 units). Prior to bradykinin administration the responses of all units to passive limb movements were recorded in order to classify the units as belonging to one of the following 4 categories: activated by non-noxious movements; weakly activated by non-noxious movements; activated only by noxious movements; not activated by movements. Bolus injections of KCl were used to test the accessibility of the units via the blood vessels. Such injections elicited a rapid burst of impulses at short latencies of less than 1 s. If this discharge did not occur, no test with bradykinin was carried out. There was no difference in latency and time course between such discharges in group III and group IV units. With only 3 exceptions the 40 units excited by KCl were also activated by bradykinin which was applied in doses from 0.026 to 26 micrograms. Higher doses were not used. For most group III and IV units the minimal effective dose of bradykinin for a clear excitation was usually either 0.26 or 2.6 micrograms. In both groups of units the bradykinin-evoked discharge generally had a uniform latency and a time course with a total duration well under 1 min. In the course of repetitive injections at intervals of 3-5 min, the latency of the evoked discharge increased gradually and its magnitude became successively smaller. This tachyphylaxis was usually very pronounced, regardless of whether low or high doses of bradykinin were administered. No differences in the bradykinin sensitivity were found between units with very low to very high thresholds to local mechanical stimulation (tested with von Frey hairs) and between units belonging to the 4 different categories of response behavior to passive innocuous and noxious joint movements. These results indicate that the sensitivity to bradykinin is shared by all fine articular afferent units, regardless of their thresholds to local mechanical stimulation and joint movement and, hence, their functional role in signaling innocuous or noxious mechanical events at the knee joint.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synaptic interactions between GABA-immunoreactive profiles and the terminals of functionally defined myelinated nociceptors in the monkey and cat spinal cord.

This study analyzes the synaptic interactions between the central terminals of A delta high threshold mechanoreceptors (A delta HTMs) and GABA-immunoreactive profiles. A delta HTM primary afferents from three monkeys and one cat were electrophysiologically identified and intracellularly labeled with HRP, and their terminal arborizations in laminae I and II of the sacrocaudal spinal cord were studied at the ultrastructural level. GABA-immunoreactive profiles in relation to A delta HTM terminals were demonstrated using postembedding colloidal gold techniques. Monkey A delta HTM terminals (n = 131) usually constituted the central element of synaptic glomeruli; they established large asymmetric synaptic contacts with 1-13 dendrites (modal value 2-4) and were surrounded by 0-6 peripheral axon terminals (modal value 2-3). The large majority (around 85%) of the peripheral axon terminals were GABA immunoreactive. They were found presynaptic to the A delta HTM terminal and/or to dendrites postsynaptic to the primary afferent terminal. Furthermore, all peripheral axon terminals found presynaptic to the A delta HTM terminals showed GABA immunoreactivity. Within a single A delta HTM fiber, this synaptic arrangement was found in 20-60% of its boutons. In addition, 28% of the postsynaptic dendritic profiles displayed weak GABA immunoreactivity. Some of them contained vesicles; however, only in a few cases did we observe synapses between a GABA-immunoreactive vesicle-containing dendrite and a dendritic profile postsynaptic to an A delta HTM terminal. Similar synaptology and interactions with GABA-immunoreactive profiles were displayed by the terminals of the single cat A delta HTM fiber studied. Our data support the hypothesis that GABA-containing neurons use both presynaptic and/or postsynaptic mechanisms to exert a powerful control, presumably inhibitory, over the transmission of nociceptive information between A delta HTM afferents and second-order neurons in monkey and cat spinal cord. Our results also imply that GABA may be released within the synaptic glomeruli formed by A delta HTM terminals either by local dendrites or by axon terminals. We discuss the possibility that these GABAergic synapses can be driven by inputs from both primary afferents and/or descending systems to modulate the transmission of nociceptive sensory information.     

Responses of fine primary afferent nerve fibres innervating the rat knee joint to defined torque

In total 23 rat knee joint afferent units with conduction velocities of 1.2-17 m/s were recorded extracellularly during inward and outward joint rotations of defined torque using a newly developed torque-meter. There was not a linear relation between the torque and the obtained angle of the joint. The mechanical thresholds of the units ranged from 10 to 60 mNm. During 10 s of stimulation with 60 mNm they responded with 12-300 action potentials. Two mechanosensitive nerve fibres could not be activated via knee joint rotation at any torque at all. The units could be classified according to their response pattern during stimulation: (1) phasic-tonic response behaviour; (2) constant discharge rate; (3) delayed onset of activity. In conclusion, the novel torque-meter allows a precise characterisation of the mechanical threshold and the corresponding response of fine afferent joint units during knee joint rotation.     

The activation of unmyelinated or myelinated afferent fibers by brief infrared laser pulses varies with skin type

Brief infrared laser pulses were applied to 3 different skin areas in man. Reaction times indicated that A delta-fibers were activated in the dorsal and volar skin of the finger whereas only C-fibers were activated in the hairy skin of the forearm. The qualitative sensations were in line with this interpretation. We conclude that the activation of unmyelinated or myelinated afferent fiber populations with brief laser pulses varies with skin type.     

Melittin selectively activates capsaicin-sensitive primary afferent fibers

Whole bee venom (WBV)-induced pain model has been reported to be very useful for the study of pain. However, the major constituent responsible for the production of pain by WBV is not apparent. Intraplantar injection of WBV and melittin dramatically reduced mechanical threshold, and increased flinchings and paw thickness. In behavioral experiments, capsaicin pretreatment almost completely prevented WBV- and melittin-induced reduction of mechanical threshold and flinchings. Intraplantar injection of melittin increased discharge rate of dorsal horn neurons only with C fiber input from peripheral receptive field, which was completely blocked by topical application of capsaicin to sciatic nerve. These results suggest that both melittin and WBV induce nociceptive responses by selective activation of capsaicin-sensitive afferent fibers.     

Morphology of central terminations of intra-axonally stained, large, myelinated primary afferent fibers from facial skin in the rat

Horseradish peroxidase was intra-axonally injected into functionally identified primary afferent fibers within the rat spinal trigeminal tract in order to study the morphology of their central terminations. They were physiologically determined to be large, myelinated, cutaneous primary afferents by means of electrical and mechanical stimulation of their receptive fields. Ninety-three axons that innervated vibrissa follicles, guard hair follicles, and slowly adapting receptors were stained for distances of 4-12 mm at the levels of the main sensory nucleus, spinal trigeminal nucleus, and rostral cervical spinal cord. The collaterals of single axons from these receptors formed terminal arbors in the outer part of the spinal trigeminal nucleus rostral to and near the level of the obex (rostral type collaterals). In the rostral part of the subnucleus caudalis (Vc) they were confined to lamina V (caudalis type collaterals) and in the caudal part of Vc and in cervical segments they were confined to lamina III/IV (spinal-dorsal-horn-type collaterals). There were no transitional forms between the rostral and caudalis types, but there was a transitional form between the caudalis and spinal dorsal horn types. This transitional form was distributed in laminae III/IV and V. The terminal arbors of the rostral type of collaterals formed an interrupted, rostrocaudally oriented column like those seen in the lumbar dorsal horn, but the column shifted down to lamina V near the obex, and more caudally, gradually shifted upward to lamina III. Major morphological differences were not observed among the three different functional types of collaterals with respect to the rostrocaudal distribution of collaterals, and the shape and location of collaterals. The differential laminar distribution of collateral arbors of single axons along the rostrocaudal axis distinguishes the spinal trigeminal nucleus from the spinal dorsal horn where functional types of mechanoreceptive afferents form continuous or interrupted sagittal columns of terminal arbors that do not shift dorsoventrally within segments.     

Clustering of primary afferent fibers in peripheral nerve fascicles by sensory modality

The spatial organization of cutaneous afferent fibers in the cat saphenous nerve was studied by recording from functionally identified units in split filaments. It was found that within each fascicle, fibers tend to be clustered together with others of like modality; they are not randomly distributed. These results suggest that the sensory modality of primary afferent neurons is determined prior to their innervation of the skin.     

Ascending unmyelinated primary afferent fibers in the dorsal funiculus

The primary purpose of the present study is to obtain evidence as to the destination of the recently discovered unmyelinated primary afferent fibers in the mammalian dorsal funiculus. To do this rat dorsal roots were transected unilaterally from segments T8 or T9 caudally, and the numbers of axons were determined in the C3 fasciculus gracilis in normal animals and from both sides of the rhizotomied animals. In addition, C3 fasciculus gracilis counts were done in animals that had complete T6 or T10 spinal transections. The data indicate that there is an 80% loss of unmyelinated axons ipsilaterally and a 60% loss contralaterally in the fasciculus gracilis of the rhizotomied animals. These findings are interpreted as indicating that a significant fraction of the unmyelinated fibers in the fasciculus gracilis ascend, presumably to the nucleus gracilis in the brain stem, and also that a significant number of these fibers branch. We also provide evidence for contralateral myelinated primary afferent fiber projection in the fasciculus gracilis and show that the myelinated primary afferent fibers seem to be a more diverse population than the unmyelinated primary afferent fibers in the C3 fasciculus gracilis.     

Serotonin and GABA-induced depolarizations of frog primary afferent fibers

The interaction of gamma-aminobutyric acid (GABA) and serotonin (5-HT) on primary afferent terminals of the isolated frog spinal cord was investigated by sucrose gap recordings from dorsal roots. Application of 5-HT (1.0-100 microM) to the Ringer's solution significantly reduced afferent terminal depolarizations elicited by concentrations of GABA ranging from 0.1 to 1.0 mM. The reductions of GABA-depolarizations which were produced by 1.0 microM 5-HT were mimicked by the 5-HT1A agonists 8-OH-DPAT (8-hydroxy-2-(n-dipropylamino)tetralin) and ipsapirone. The effects of ipsapirone were reversed by the 5-HT1A antagonist spiperone. The decreases of GABA-depolarizations produced by high doses of 5-HT were duplicated by application of alpha-methyl-5-HT, a 5-HT1C/2 agonist and reversed by superfusion of the cord with manserin, a 5-HT1C/2 antagonist. The presumptive 5-HT1A receptor-mediated effects of 1.0 microM 5-HT and 8-OH-DPAT appeared to result from a direct action on afferent terminals because the reduction of GABA responses was unchanged by addition of TTX to the Ringer's solution. In contrast, the putative 5-HT1C/2 receptor actions of 100 microM 5-HT and alpha-methyl-5-HT were substantially reduced by TTX and are presumably caused by activation of receptors located on interneurons. GABAB receptors did not appear to be affected by addition of 5-HT at low or high concentrations because baclofen-induced afferent terminal hyperpolarizations remained unchanged during exposure to 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ectopic impulse generation and autoexcitation in single myelinated afferent fibers in patients with peripheral neuropathy and positive sensory symptoms

Microneurographic studies were performed using cutaneous nerves of 8 patients with documented peripheral neuropathy who expressed positive sensory symptoms. Intraneural recordings in single myelinated fibers revealed: (i) ectopic generation of bursts of spontaneous action potentials; (ii) ectopic generation of ongoing repetitive discharges transiently interrupted by natural stimulation of the receptive field; and (iii) repetitive discharges triggered by a preceding action potential. These results provide direct evidence of a peripheral pathophysiological basis for spontaneous and stimulus-induced paresthesias and dysesthesias in patients with peripheral neuropathy. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 1661–1667, 1998     

Conduction velocity changes along lumbar primary afferent fibers in cats

Though peripheral conduction velocity is widely used to characterize afferent fibers according to somatosensory modality, disagreement exists as to whether or not conduction velocity varies along such an axon's length. Therefore, in this experiment, conduction velocities were measured over very short axonal segments (7.5 to 15 mm) within the posterior tibial nerve, sciatic nerve, and L7 dorsal root, using the method of spike-triggered averaging of neurograms recorded from tripolar electrodes. The conduction velocities for several units were also determined using electrical stimulation, so that the accuracy of the two techniques could be compared. For most units, dorsal root and sciatic nerve conduction velocities were not significantly different; however, they were not tightly correlated. Tibial nerve conduction velocity averaged 86% of that within the sciatic nerve. Variations in sciatic nerve conduction velocity within adjacent axonal segments (9 mm in length) rarely exceeded experimental error. It appears that spike-triggered averaging of signals from tripolar electrodes separated 15 mm or more apart is a more accurate method for measuring conduction velocity than electrical stimulation, which was subject to several large errors.     

Electron microscopic observations of terminals of functionally identified afferent fibers in cat spinal cord

Using the method of intra-axonal injection of horseradish peroxidase, functionally identified afferent fibers from three slowly adapting (Type I) receptors and one Pacinian corpuscle in the glabrous skin of the hind paw of the cat were stained. Electron microscopic observation of the terminals of these fibers revealed predominantly axodendritic asymmetric synapses containing round, clear vesicles. Multiple synapses on a single dendrite were observed, separated by as little as 900 mm from one another.     

Effects of aging on myelinated nerve fibers in monkey primary visual cortex

In monkeys, myelin sheaths of the axons in the vertical bundles of nerve fibers passing through the deeper layers of primary visual cortex show age-related alterations in their structure. These alterations have been examined by comparing the myelin sheaths in young monkeys, 5-10 years old, with those in old monkeys, between 25 and 33 years of age. The age-related alterations are of four basic types. In some sheaths, there is local splitting of the major dense line to accommodate dense cytoplasm derived from the oligodendrocytes. Other sheaths balloon out, and in these locations, the intraperiod line in that part of the sheath opens up to surround a fluid-filled space. Other alterations are the formation of redundant myelin so that a sheath is too large for the enclosed axon and the formation of double sheaths in which one layer of compact myelin is surrounded by another one. These alterations in myelin increase in frequency with the ages of the monkeys, and there is a significant correlation between the breakdown of the myelin and the impairments in cognition exhibited by individual monkeys. This correlation also holds even when the old monkeys, 25 to 33 years of age, are considered as a group. It is suggested that the correlation between the breakdown of myelin in the old monkeys and their impairments in cognition has not to do specifically with visual function but to the role of myelin in axonal conduction throughout the brain. The breakdown of myelin could impair cognition by leading to a change in the conduction rates along axons, resulting in a loss of synchrony in cortical neuronal circuits.     

Morphometric alteration of rat myelinated fibers with aging

The number, size, density and pathologic alterations of myelinated fibers (MF) of ventral and dorsal roots and of peroneal and sural nerves in groups of female Fischer 344 rats at 10, 20, and 30 months of age were evaluated to characterize nerve changes with old age. Except for minimal changes in the peroneal nerve, no statistically significant alteration in number of MF/nerve or in the fascicular area was associated with aging. The unaltered number was misleading since striking changes in MF size distribution, pathologic alterations of fibers and the presence of regeneration clusters suggested age-related degenerative and regenerative events. These changes were most dramatic in the ventral root, where myelin infolding, myelin separation from axon and ballooning, macrophagia and hyperplasia of Schwann cell nuclei were pronounced. Concomitant with these alterations, axonal atrophy (a reduction in caliber of axons relative to myelin spiral length or number of myelin lamellae) was demonstrated in MF of the ventral root in old age.     

Synaptic transfer from outer hair cells to type II afferent fibers in the rat cochlea

Type II cochlear afferents receive glutamatergic synaptic excitation from outer hair cells (OHCs) in the rat cochlea. However, it remains uncertain whether this connection is capable of providing auditory information to the brain. The functional efficacy of this connection depends in part on the number of presynaptic OHCs, their probability of transmitter release, and the effective electrical distance for spatial summation in the type II fiber. The present work addresses these questions using whole-cell recordings from the spiral process of type II afferents that run below OHCs in the apical turn of young (5-9 d postnatal) rat cochlea. A "high potassium puffer" was used to elicit calcium action potentials from individual OHCs and thereby show that the average probability of transmitter release was 0.26 (range 0.02-0.73). Electron microscopy showed relatively few vesicles tethered to ribbons in equivalent OHCs. A "receptive field" map for individual type II fibers was constructed by successively puffing onto OHCs along the cochlear spiral, up to 180 μm from the recording pipette. These revealed a conservative estimate of 7 presynaptic OHCs per type II fiber (range 1-11). EPSCs evoked from presynaptic OHCs separated by >100 μm did not differ in amplitude or waveform, implying that the type II fiber's length constant exceeded the length of the synaptic input zone. Together these data suggest that type II fibers could communicate centrally by maximal activation of their entire pool of presynaptic OHCs.     

Primary afferent terminals that express presynaptic NR1 in rats are mainly from myelinated,mechanosensitive fibers

Presynaptic N-methyl-D-aspartate (NMDA) receptors in terminals of primary afferents to spinal cord of rats were first reported by Liu et al. (1994; Proc. Natl. Acad. Sci. USA 91:8383-8387) and were proposed to modulate nociceptive input (Liu et al. [1997] Nature 386:721-724). We previously demonstrated kainate and AMPA receptors in numerous primary afferent terminals in the spinal cord fixed with diluted paraformaldehyde and no glutaraldehyde. Therefore, we reinvestigated the occurrence of presynaptic NMDAR1 (NR1) with this fixation protocol. With confocal microscopy, numerous immunofluorescent puncta were double-stained for NR1 and the presynaptic marker synaptophysin throughout the spinal gray. NR1-immunostained puncta costained more frequently with a tracer that labels myelinated afferents (cholera toxin subunit B; CTB) than with a tracer that labels non-peptidergic unmyelinated afferents (Griffonia simplicifolia isolectin B4; IB4). Virtually no double staining was found for NR1 and calcitonin gene-related peptide (CGRP), which labels somatic peptidergic primary afferents. In the gracile nucleus, virtually all puncta labeled for CTB appeared immunopositive for NR1. At the electron microscopic level, most immunopositive terminals in spinal cord and gracile nucleus displayed morphological characteristics of endings of myelinated primary afferents. NR1 was presynaptic in 60-65% of all synapses in which it was expressed pre- or postsynaptically, or both, in spinal laminae I-IV. Estimates for the gracile nucleus were higher (80%). No presynaptic NR1 was found in the ventroposterior thalamus. Because of the relative sparsity of presynaptic NR1 in terminals in laminae I and IIo and in terminals of peptidergic unmyelinated afferents, it is suggested that presynaptic NMDA receptors play a more significant role in modulation of mechanosensitive, innocuous input than in nociception.     

Number and diameter distribution of myelinated afferent fibers innervating the paws of the cat and monkey.

The number and the diameter distribution of the myelinated cutaneous fibers innervating fore and hind paw were histologically examined in the cat and monkey. In five cats, the superficial peroneal nerve innervating the dorsal surface of the hind paw and the superficial plantar nerve innervating the palmar surface were composed of 2668–2950 fibers, 40–44% of which were group II fibers. On the other hand, the superficial radial nerve innervating the dorsal surface of the fore paw and the sensory branch of median nerve innervating the palmar surface were composed of 3270–3680 fibers, of which 67–72% were group II fibers. Therefore, it was found that the cutaneous fore paw nerves contained more group II fibers than the hind paw cutaneous nerves. In five monkeys the percentages of group II fibers composing the superficial peroneal nerve and superficial plantar nerve (2893–3374 fibers) were the same as those of the hind paw nerves in the cat. On the other hand, the percentages of group II fibers of the median nerve (4173–4472 fibers) were 75–78%, about 10% larger than those of the superficial radial nerve (3596–3821 fibers) whose values were 66–69%. Therefore, in the monkey, the forepaw nerves innervating the palmar surface also contain more group II afferent fibers compared to the ones innervating the dorsal one (hairy skin).