Microtubules and calibers in normal and regenerating axons of the sural nerve of the rat

The calibers and microtubular content of axons were studied in normal and regenerating fibers of the sural nerve from 17 to 122 days after a lesion of the sciatic nerve of young adult rats. During this period (70-175 days of age), the cross-sectional area of control myelinated axons almost doubled but that of nonmedullated axons did not change. In regenerating nerves, after 122 days of recovery, the cross-sectional area of myelinated fibers was still 38% below that of the normal side. In contrast, the regenerating nonmedullated population was richer in fine (less than 0.2 micron2) and in coarse (greater than 0.9 micron2) fibers than on the control side; the cross-sectional area averages were 0.50 and 0.54-0.70 micron 2 for the normal and regenerating populations, respectively. The microtubular density of normal 3-micron myelinated fibers averaged 24.0 microtubules/micron2. In regenerating fibers of the same size the density varied between 19.2 and 23.2 microtubules/micron2. Microtubular density values of normal and regenerating fibers were not statistically different. In nonmedullated fibers, the microtubular content (expressed as microtubular density or number of microtubules per axon) correlated with the caliber of the fiber. In these correlations, only minor differences were observed between regenerating and uninjured fibers. Our results indicate that nonmedullated fibers terminate their radial growth well before myelinated fibers do, and that axonal microtubular content correlates with the local size of the fiber and is largely insensitive to regeneration.     

Calcitonin gene-related peptide immunostained axons provide evidence for fine primary afferent fibers in the dorsal and dorsolateral funiculi of the rat spinal cord

The hypothesis being tested in the present paper is that there are large numbers of fine primary afferent axons in the dorsal and dorsolateral funiculi of the lumbar spinal cord of the rat. The data show numerous calcitonin gene-related peptide labeled fine myelinated and unmyelinated axons in these funiculi. Approximately 95% of the labeled axons disappear after dorsal rhizotomy. Accordingly, the hypothesis is confirmed. Thus it is becoming apparent that fine primary afferent fibers are more widely distributed in spinal white matter than had been previously recognized. Implications are that it is not possible to find areas in the spinal white matter that contain only large myelinated sensory axons and that significant numbers of fine primary afferent fibers will be lost even if lesions are restricted to the dorsal funiculus. The sizable population of fine myelinated primary afferent axons in the dorsal funiculus is emphasized. An obvious question, suggested by significant differences in average diameters of the axons in the different pathways, is whether there are differences in the types of information carried by the fine afferent fibers in their different locations in the white matter of the lumbar cord.     

Unmyelinated primary afferent fibers in dorsal funiculi of cat sacral spinal cord

The present study tests the hypothesis that there are numerous unmyelinated primary afferent fibers in cat posterior funiculi. The animals have unilateral dorsal rhizotomies from L6 to Ca3. One week later the axons of both S2 dorsal funiculi are counted. The data indicate that there are approximately 22,500 myelinated and 8,500 unmyelinated axons on the unoperated side and 11,000 myelinated and 3,900 unmyelinated axons on the operated side. On this basis, we suggest that 51% of the myelinated and 54% of the unmyelinated axons in cat dorsal funiculi arise from dorsal root ganglion cells and thus are primary afferent axons. If this is correct, then 71% of the primary afferent axons in the cat dorsal funiculus are myelinated and 29% are unmyelinated. The function of this large group of previously unsuspected fine sensory axons remains to be determined.     

Analgesic effect of intrathecal morphine demonstrated in ascending nociceptive activity in the rat spinal cord and ineffectiveness of caerulein and cholecystokinin octapeptide

The effect of intrathecal injections of morphine and the two peptides, caeruelin and cholecystokinin octapeptide (CCK-8), on the activity in ascending axons of the spinal cord evoked by electrical stimulation of primary nociceptive afferents was studied in spinal rats with decerebration. Morphine (20 μg) depressed the spontaneous activity and the activity evoked from either Aδ- or C-fibres. The co-activation by Aδ-fibre stimulation of ascending axons activated by stimulation of C-fibres and the activity in ascending axons activated by stimulation of afferent Aβ-fibres were not influenced by morphine. C-Fibre-evoked ascending activity was also depressed by morphine (10 μg and 5 μg). Ascending nociceptive activity was not changed by caerulein (30 ng) and CCK-8 300 ng, but it was depressed by a subsequent injection of morphine (20 μg). The depressant effects of morphine were abolished by an intravenous injection of naloxone which, when given alone, facilitated the ascending nociceptive activity. It is concluded that: (1) an intrathecal injection of morphine selectivity depressed the ascending nociceptive activity; (ii) the depression produced by morphine is an equivalent for spinal analgesia following intrathecal injection of morphine to man; and (iii) the two components of the spinal nociceptive system, the motor and the sensory path, can independently be influenced by drugs.     

Fine afferent fibers from viscera do not terminate in the substantia gelatinosa of the thoracic spinal cord

Transganglionic transport of horseradish peroxidase (HRP) has been used to study the anatomy of the central projection of somatic and visceral afferent fibers to the thoracic spinal cord of the cat. A dense concentration of somatic afferent fibers and terminals was found in laminae I and II of the dorsal horn and more scattered terminals were present in laminae III, IV and V and in Clarke's column. In contrast, visceral afferent fibers and terminals were found only in lamina I or reaching lamina V via a small bundle of fibers located in the lateral border of the dorsal horn. These results indicate that fine afferent fibers from viscera, unlike those of cutaneous origin, do not project to the substantia gelatinosa (lamina II) of the dorsal horn.     

The distribution with the spinal cord of visceral primary afferent axons carried by the lumbar colonic nerve of the cat

Horseradish peroxidase taken up by the sensory axons in the lumbar colonic nerves in 5 cats was observed in the dorsal root ganglia and in the spinal cord in segments L1 through L5. Reaction product was observed in Lissauer's tract, the dorsal columns and laminae I, V, VII and X in a pattern typical of visceral primary afferents from other nerves. A small number of preganglionic neurons were also labeled.     

Recurrent potentials in human peripheral sensory nerve: possible evidence of primary afferent depolarization of the spinal cord.

To study slowly conducted components of the orthodromic compound sensory action potential (CSAP), the response evoked at the lateral malleolus in the sural nerve was recorded through near-nerve needles at two to four sites along the nerve at midcalf. When 500 to 2000 responses were averaged at high gain, components with latencies of 30 to 80 ms were often recorded. In contrast to the main component and late components with latencies of less than 15 to 20 ms, the latencies of these extremely late components diminished the closer to the spinal cord that they were recorded. This suggested that the components were conducted antidromically from proximal to distal. This assumption was supported by abolishing the components by local anesthesia of the nerve proximal to the recording electrodes. These antidromic potentials therefore appear to be due to recurrent discharges in the sural nerve. Recurrent discharges were recorded from 65% of 60 subjects (18 normal subjects and 42 patients with peripheral or central nervous system disorders). The latencies of the recurrent discharges allowed conduction to and back from the spinal cord. Although the origin of these potentials remains unknown, we suggest that they are due to dorsal root reflexes within the spinal cord. In this case, the responses may be a direct expression of primary afferent depolarization (PAD) seen in presynaptic inhibition, and may be of value in further studies on the physiology and pathophysiology of presynaptic inhibition of cutaneous fibers in man.     

Alterations in primary afferent input to substantia gelatinosa of adult rat spinal cord after neonatal capsaicin treatment

Primary afferent fibers are divided into three main subgroups: Abeta-, Adelta-, and C-fibers. Morphological studies have demonstrated that neonatal capsaicin treatment (NCT) depletes C-fiber inputs in the spinal dorsal horn; the electrophysiological features of the NCT-induced changes, however, remain unclear. This issue was addressed by performing whole-cell voltage-clamp recordings from substantia gelatinosa (SG) neurons in dorsal root-attached spinal cord slices. When estimated from excitatory postsynaptic currents (EPSCs) evoked by stimulating primary afferent fibers, 24 (49%) of 49 neurons examined exhibited C-fiber EPSCs that were either monosynaptic (n = 15) or polysynaptic (n = 9) in origin; only two of the neurons had Abeta-fiber responses. In NCT rats, however, SG neurons exhibiting C-fiber-mediated EPSCs decreased to 7% (3 of 41 neurons tested), whereas Abeta-fiber EPSCs were observed in 21 (51%) of the neurons, and 14 (67%) of them exhibited monosynaptic ones. There was no change in the cell proportion having Adelta-fiber innervation after NCT. Our electrophysiological data suggest that NCT diminishes primary afferent C-fiber inputs while enhancing Abeta-fiber direct innervation in the SG in adulthood.     

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 vagal afferent nerve stimulation on noxious heat-evoked Fos-like immunoreactivity in the rat lumbar spinal cord

Electrophysiological and behavioral studies have described modulation of nociception by vagal afferent fibers. The objectives of this study were to (1) use Fos-like immunoreactivity as a marker for neuronal activity to examine populations of neurons in the spinal cord that are activated by a noxious heat stimulus, (2) determine whether heat-evoked Fos-like immunoreactivity can be modulated by vagal afferent stimulation, and (3) determine whether vagallymediated effects on heat-evoked Fos-like immunoreactivity can be blocked by intrathecally administered serotoninergic receptor and α-adrenergic receptor antagonists. Neurons demonstratiring Fos-like immunoreactivity were located in the ipsilateral superficial and deep dorsal horn laminae extending from the caudal L₃ through the rostral L₆ region of the spinal cord. Stimulation of the right cervical vagus nerve attenuated significantly (42%) heat-evoked Fos-like immunoreactivity in the superficial laminae. The reduction in Fos-like immunoreactivity by vagal stimulation was abolished by intrathecal administration of methysergide, a nonselective serotoninergic receptor antagonist, but not by phentolamine, a nonselective α-adrenoceptdr antagonist. These results suggest that vagal afferent modulation of spinal nociceptive transmission is mediated, at least in part, by serotonin receptors. © 1994 Wiley-Liss, Inc.     

Axonal guidance of adenosine deaminase immunoreactive primary afferent fibers in developing mouse spinal cord

This study examined the precision of central fiber growth in a subpopulation of dorsal root ganglion neurons in developing mouse spinal cord. Immunohistochemical techniques using a monospecific, polyclonal antiserum to mouse adenosine deaminase (ADA) were utilized to label a population of primary sensory afferents that have been found to exclusively innervate laminae I and II of the dorsal horn in adult mice. Initial growth of ADA-immunoreactive (ADA-IR) primary afferents occurred very early in development, embryonic day 10 (E10), a time coincident with the earliest settling time of dorsal root ganglion neurons. Adenosine deaminase immunoreactive primary afferents were observed throughout the cross-sectional area of the primordial dorsal funiculus (DF) as early as E10. Immunostained fibers remained quiescent in the DF during its growth and separation into the tract of Lissauer and dorsal column pathway. By E15, the two pathways had formed and ADA-IR fibers were observed exclusively in the tract of Lissauer. This segregation of fibers remained throughout development and reflected the adult pattern. Growth was reinitiated at E16 when the fibers advanced into the dorsal horn and proceeded directly to laminae I and II mimicking their adult distribution. Exuberant fiber growth was not detected throughout their development. These results strongly suggest that ADA-IR fibers exhibit precise fiber guidance to a preferred pathway, the tract of Lissauer, and accurate laminar innervation of the dorsal horn.     

Myelination of the ventral and dorsal roots of the C8 and L4 segments of the spinal cord at different stages of development in the gray opossum,Monodelphis domestica

We have quantified the number and size of myelinated fibers of the ventral and dorsal roots of selected segments that innervate the forelimbs (C8) and hindlimbs (L4) in the developing opossum, Monodelphis domestica. The gray opossum was chosen because it is born very immature and its somatomotor development occurs almost entirely postnatally. After aldehyde fixation, osmium postfixation, and resin embedding, the roots were cut transversely (1.5 μm), stained with toluidine blue, and observed and photographed by using light microscopy. The counts and measurements were made with a digitizing table. Myelination of the C8 and L4 roots begins during the second week of life and occurs according to two gradients: rostrocaudal and ventrodorsal. The number of myelinated fibers in these roots increased over approximately 7 weeks after which an excess, compared with their adult value, was recorded during the following weeks in three of the four roots. The supernumerary myelinated fibers are presumed to be collaterals. The fibers increased in diameter until at least 98 days. The classification according to size for the ventral roots (alpha and gamma) became evident in the fourth week, but that in types I, II, and III for the dorsal roots was never clear. There was no significant difference in the number and size distribution of myelinated fibers between sexes until late in development. The fibers innervating the limbs thus become myelinated postnatally in the opossum, a process that occurs over a protracted period and that continues after sensorimotor reflexes and locomotion appear adult-like. J. Comp. Neurol. 386:203–216, 1997. © 1997 Wiley-Liss, Inc.     

GABA and glycine-like immunoreactivity at axoaxonic synapses on 1a muscle afferent terminals in the spinal cord of the rat

The object of this study was to analyze the synaptic interactions of identified muscle spindle afferent axon terminals in the spinal cord of the rat. Group 1a muscle afferents supplying the gastrocnemius muscle were impaled with microelectrodes in the dorsal white matter of the spinal cord and stained by intracellular injection with Neurobiotin. Postembedding immunogold techniques were used to reveal GABA- and glycine-like immunoreactivity in boutons presynaptic to afferent terminals in the ventral horn and the deep layers of the dorsal horn. Serial-section reconstruction was used to reveal the distribution of synaptic contacts of different types on the afferent terminals. The majority of afferent boutons received axoaxonic and made axodendritic or axosomatic synaptic contacts. In the ventral horn, 91% of boutons presynaptic to the afferent terminals were immunoreactive for GABA alone and 9% were immunoreactive for both GABA and glycine. The mean number of axo-axonic contacts received per terminal was 2.7, and the mean number of synaptic contacts at which the terminal was the presynaptic element was 1.4. In the deep layers of the dorsal horn, 58% of boutons presynaptic to afferent terminals were immunoreactive for GABA alone, 31% were immunoreactive for GABA and glycine, and 11% for glycine alone. The mean number of axoaxonic contacts received per afferent terminal in this region was 1.6 and the mean number of synaptic contacts at which the terminal was the presynaptic element was 0.86. This clearly establishes the principle that activity in 1a afferents is modulated by several neurochemically distinct populations of presynaptic neuron.     

Somatosensory evoked potentials induced by stimulating a variable number of nerve fibers in rat

Somatosensory evoked potentials (SEPs) were recorded from rat spinal cord (sSEPs) and cerebral cortex (cSEPs). Stimulus sites included either one or both sural nerve branches having different fiber populations (group A), or distal to a lesion of controlled size of the sural nerve made 1 week earlier (group B). In the two groups of animals, amplitudes of SEPs correlated with the quantity of large myelinated nerve fibers. Peak latencies of sSEPs in group A related to the ratio of sizes of transmitting fibers. sSEPs and cSEPs in both groups A and B could be recorded in a reproducible fashion by stimulating sural nerve branches or lesioned nerve trunks containing only 100 or less nerve fibers greater than 4 m?m in size. Thus, presence of sSEPs or cSEPs after stimulation distal to a lesion site does not insure that many nerve fibers have continuity with the central nervous system (CNS). © 1993 John Wiley & Sons, Inc.     

An electron microscopic study of terminals of rapidly adapting mechanoreceptive afferent fibers in the cat spinal cord

The intra-axonal horseradish peroxidase technique was used to examine the central terminals of 7 A beta primary afferent fibers from rapidly adapting (RA) mechanoreceptors in the glabrous skin of the cat's hindpaw. At the light microscopic level, labelled collaterals were seen to bear occasional boutonlike swellings, mostly (75-82%) of the en passant type. These swellings were distributed more or less uniformly from lamina III to a dorsal part of lamina VI in the dorsal horn, over a maximum longitudinal extent of about 4 mm. At the electron microscopic level, we observed that labelled boutons of RA afferent fibers were 1.0 to 3.3 micrometers in longest sectional dimension, and contained clear, round synaptic vesicles. They frequently formed asymmetric axospinous and axodendritic synapses and commonly appeared to receive contacts from unlabelled structures containing flattened or pleomorphic vesicles plus occasional large dense-cored vesicles. The examination of synaptic connectivity over the entire surface of individual boutons indicated that RA afferent boutons each made contacts with an average of one spine and one dendrite and, in addition, appeared to be postsynaptic to an average of two unlabelled vesicle-containing structures. This synaptic organization was, in general, more complex than that we had seen previously in Pacinian corpuscle (PC) and slowly adapting (SA) type I mechanoreceptive afferent fibers. Our findings indicate that RA, SA, and PC afferent terminals, while displaying some differential synaptic organizations, have many morphological and synaptological characteristics in common. These afferent terminals, in turn, seem to be generally distinguishable from the terminals of muscle spindle Ia afferents or unmyelinated primary afferents.     

The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord

Transganglionic transport of wheatgerm agglutinin conjugated horse-radish peroxidase (WGA-HRP) was used to reveal the central distribution of terminals of primary afferent fibers from peripheral nerves innervating the hind leg of the rat. In separate experiments the sizes and locations of cutaneous peripheral receptive fields were determined by electrophysiological recording techniques for each of the nerves that had been labeled with WGA-HRP. By using digital image analysis, the sizes and positions of the peripheral receptive fields were correlated with the areas of superficial dorsal horn occupied by terminals of primary afferents from each of these receptive fields. Data were obtained from the posterior cutaneous nerve of the thigh, lateral sural, sural, saphenous, superficial peroneal, and tibial nerves. The subdivisions of the sciatic nerve, the sural, lateral sural, superficial peroneal, and tibial nerves each projected to a separate and distinct region of the superficial dorsal horn and collectively formed a "U"-shaped zone of terminal labeling extending from lumbar spinal segments L2 to the caudal portions of L5. The gap in the "U" extended from L2 to the L3-4 boundary and was occupied by terminals from the saphenous nerve. Collectively, all primary afferents supplying the hindlimb occupied the medial 3/4 of the superficial dorsal horn with terminals from the tibial nerve lying most medially and occupying the largest of all the terminal fields. Afferents from the superficial peroneal lay in a zone between the medially situated tibial zone and the more laterally placed sural zone. Afferents from the posterior cutaneous nerve were located most caudally and laterally. Terminal fields from the posterior cutaneous and saphenous nerves differed from the others in having split representations caused presumably by their proximity to the mid-axial line of the limb. Comparisons between the peripheral and the central representations of each nerve revealed that 1 mm2 of surface area of the superficial dorsal horn serves approximately 600-900 mm2 of hairy skin and roughly 300 mm2 of glabrous skin. The vast majority of terminal labeling observed in the dorsal horn was found in the marginal layer and substantia gelatinosa, suggesting that small diameter afferents have an orderly somatotopic arrangement in which each portion of the skin surface is innervated by afferent fibers that terminate in preferred localities within the dorsal horn.     

Mechanical changes in the amphibian spinal cord produced by afferent volleys of nerve impulses

A volley of impulses entering the toad spinal cord via large myelinated fibers in the lumbar dorsal roots was shown to evoke a contraction of the cord, which lasted for about 100 ms or more. A volley entering the cord antidromically via the ventral roots produced only a small, brief contraction. When two electric shocks were delivered to the same dorsal roots at a short interval, the contraction associated with the second shock was small; a period of about 1 s was required for a full recovery. When two shocks were applied separately to two neighboring dorsal roots, the contraction associated with the second shock was partially or totally occluded. Electric polarization of the dorsal root fibers produced mechanical changes in the cord. The effects of magnesium salt, GABA, glutamate and several other neuropharmacological agents on the contractile process were investigated. The experimental findings suggest that the contractile process is related to the phenomenon of primary afferent depolarization.     

Effect of stimulation in the periaqueductal grey matter on activity in ascending axons of the rat spinal cord: selective inhibition of activity evoked by afferent Aδ and C fibre stimulation and failure of naloxone to reduce inhibition

In rats with prenigral decerebration, the effect was studied of electrical stimulation of the periaqueductal grey matter (PAG) on the activity recorded from axons ascending in the spinal cord. These axons were activated by electrical stimulation of afferent Aβ, Aδ and C fibres in the ipsilateral sural nerve.Stimulation of the PAG with trains of impulses by itself evoked ascending activity, but strongly depressed the impulse transmission from C fibres to neurones with ascending axons. It exerted a weaker effect on impulse transmission from Aδ fibres and had no effect on impulse transmission from Aβ fibres to neurones with ascending axons. Intravenous naloxone, 1 mg/kg, did not diminish the depressant effect of PAG stimulation.Intravenous morphine depressed the activation of ascending axons from afferent C fibres (0.5 mg/kg) more markedly than that from afferent Aδ fibres (2 mg/kg), but did not modify the depression of ascending activity produced by PAG stimulation. Naloxone antagonized the depressant effect of morphine.The results indicate that PAG stimulation inhibits ascending activity evoked by noxious stimuli by a mechanism which does not necessarily involve endogenous opiates.     

Serotonergic modulation of spinal ascending activity and sacral reflex activity evoked by pelvic nerve stimulation in cats

Serotonin (5-HT) may be inhibitory to micturition at a spinal level. A potential mechanism of action for serotonergic inhibition of bladder function is a depression of the ascending limb of the supraspinal reflex mediating micturition. Ascending activity evoked by pelvic nerve stimulation was recorded in the thoracic spinal cord of anesthetized cats. For comparison, spinal reflex activity evoked by pelvic nerve stimulation was recorded on the pudendal nerve. The effects of intrathecal administration of serotonergic agents were examined to determine whether spinal and supraspinal responses to bladder afferent activation were modulated by 5-HT. Methysergide (60 nmol), a non-selective serotonergic antagonist, increased ascending activity by 61±7% and depressed spinal reflex activity by 38±6%. Zatosetron (10 nmol), a 5-HT₃ antagonist had a similar effect on both activities (increased by 93±24% and decreased by 77±7%, respectively). The effect on ascending activity of blocking 5-HT₃ receptors was also confirmed with ICS 205930 and MDL 72222. 2-Methyl-5-HT (800 nmol), a 5-HT₃ agonist, depressed ascending activity to 46±9% of control, but enhanced spinal reflex activity by 73±92%. These results demonstrate that stimulation of 5-HT₃ and methysergide-sensitive 5-HT receptors can inhibit ascending activity and facilitate spinal reflex activity elicited by activation of bladder afferents. It is suggested that descending serotonergic pathways may participate in the spinal coordination of urinary continence.     

A-fiber sensory input induces neuronal cell death in the dorsal horn of the adult rat spinal cord.

Excitotoxicity due to excessive synaptic glutamate release is featured in many neurological conditions in which neuronal death occurs. Whether activation of primary sensory pathways can ever produce sufficient over-activity in secondary sensory neurons in the dorsal horn of the spinal cord to induce cell death, however, has not been determined. In this study, we asked whether activity in myelinated afferents (A fibers), which use glutamate as a transmitter, can induce cell death in the dorsal horn. Using stereological estimates of neuron numbers from electron microscopic sections, we found that stimulation of A-fibers in an intact sciatic nerve at 10 Hz, 20 Hz, and 50 Hz in 10-minute intervals at a stimulus strength that activates both Abeta and Adelta fibers resulted in the loss of 25% of neurons in lamina III, the major site of termination of large Abeta fibers, but not in lamina I, where Adelta fibers terminate. Furthermore, sciatic nerve lesions did not result in detectable neuron loss, but activation of A fibers in a previously sectioned sciatic nerve did cause substantial cell death not only in lamina III but also in laminae I and II. The expansion of the territory of A-fiber afferent-evoked cell death is likely to reflect the sprouting of the fibers into these laminae after peripheral nerve injury. The data show, therefore, that primary afferent A-fiber activity can cause neuronal cell death in the dorsal horn with an anatomical distribution that depends on whether intact or injured fibers are activated. Stimulation-induced cell death potentially may contribute to the development of persistent pain.