Unmyelinated Primary Afferent Fibers Research Articles

The Peripheral Role of CCL2 in the Anti-Nociceptive Effect of Sigma-1 Receptor Antagonist BD1047 on Inflammatory Hyperalgesia in Rats.

Our recent study demonstrated that the CC-chemokine ligand 2 (CCL2) present in primary afferent fibers (PAFs) plays an important role in the microglia-dependent neuronal activation associated with zymosan-induced inflammatory pain. The present study was aimed to evaluate whether BD1047 (a prototypical sigma-1 receptor (Sig-1R) antagonist) is capable of modifying elevated levels of inflammation-evoked CCL2 as a peripheral antinociceptive mechanism. In DRG primary culture, zymosan dose-dependently increased CCL2 release from isolectin B4 (IB4)-positive DRG neurons, a process that was inhibited by co-culture with BD1047. Single treatment of BD1047 before intraplantar injection of zymosan in rats significantly reduced thermal hyperalgesia and mechanical hyperalgesia, as well as CCL2 expression in DRG neurons and microglia activation in the spinal dorsal horn. In the Complete Freund’s adjuvant (CFA)-induced inflammation model, repeated administration of BD1047 dramatically attenuated thermal hyperalgesia and mechanical hyperalgesia, and significantly diminished CCL2 immunoreactivity and microglia activation. Notably, CFA-induced inflammation significantly increased Sig-1R immunoreactivity in DRG neurons, which was co-localized with CCL2 and IB4, respectively. Taken together, our results suggest that BD1047′s anti-nociceptive property was substantially mediated by the inhibition of CCL2 release in unmyelinated PAFs and that this may, in turn, have attenuated the spinal microglia activation that is associated with inflammatory pain.

The CCL2 elevation in primary afferent fibers produces zymosan-induced hyperalgesia through microglia-mediated neuronal activation in the spinal dorsal horn

Although C–C motif chemokine ligand 2 (CCL2) plays a critical role in the pathogenesis of neuropathic pain through neuron-microglia interactions, its pronociceptive function underlying inflammatory pain remains to be fully understood. The present study aimed to elucidate the potential role of CCL2 in pain sensitization following zymosan-induced inflammation. Intraplantar injection of zymosan into the rat hind paw significantly increased the expression of CCL2 in both dorsal root ganglions and the superficial dorsal horn. The expression of CCL2 was exclusively present in the isolectin B4-positive unmyelinated primary afferent fibers, but no in other cells of the spinal cord. Intrathecal administration of RS504393 (a CCR2 antagonist) markedly reduced the zymosan-induced thermal and mechanical hyperalgesia accompanied with reduced expression in the spinal cord of c-Fos, CD11b, phosphorylated p38 mitogen activated protein kinases (p-p38), and interleukin-1β. Whole cell patch-clamp recordings on spinal cord slices further revealed that the incubation of CCL2 evoked an evident inward current in substantia gelatinosa neurons and increased level of p-p38 in microglia. Moreover, co-incubation with minocycline (an inhibitor of microglial activation) prevented CCL2-mediated activation in the spinal cord slice. Taken together, we propose that the increased CCL2 expression from primary afferent fibers following zymosan-induced inflammation activates nociceptive neurons in the spinal dorsal horn via a microglia-dependent mechanism.

The spinal antinociceptive mechanism determined by systemic administration of BD1047 in zymosan-induced hyperalgesia in rats.

Although sigma-1 receptor (Sig-1R) antagonists have a potential antinociceptive effect in inflammatory diseases, the precise mechanism is not fully understood. The present study was aimed to elucidate the role of spinal neurons and microglia in the anti-nociceptive mechanism of BD1047 (a prototypical Sig-1R antagonist) using an inflammatory pain model based on intraplantar injection of zymosan. Oral pretreatment with BD1047 dose-dependently reduced zymosan-induced thermal and mechanical hyperalgesia as well as spinal neuronal activation including increased immunoreactivity of Fos, protein kinase C (PKC) and 'PKC-dependent phosphorylation of the NMDA receptor subunit 1' (pNR1). Zymosan also led to increased CD11b immunoreactivity (a marker of microglia) accompanied by 'phosphorylated p38 mitogen activated protein kinase' (p-p38MAPK) and interleukin-1βimmunoreactivity in the spinal dorsal horn. Intrathecal injection of a microglia modulator (minocycline), p38MAPK inhibitor (SB203580) or interleukin-1βneutralizing antibody significantly attenuated zymosan-induced hyperalgesia. Specifically, oral pretreatment with BD1047 reduced the immunoreactivity of CD11b, p-p38MAPK and interleukin-1β. In the spinal cord section, Sig-1R immunoreactivity was exclusively distributed in both spinal dorsal horn neurons and central endings of unmyelinated primary afferent fibers but not in glia. Intrathecal injection of BD1047 alleviated zymosan-induced hyperalgesia up to the level of oral administration. Taken together, our data imply that antinociceptive effect induced by oral treatment with BD1047 may be mediated, at least in part, by the inhibition of neuronal and microglial activation in the spinal cord triggered by inflammatory conditions.

Propagation of Spinal Nociceptive Activity in the Spatial and Temporal Domains

Nociceptive stimuli are transmitted through thinly myelinated or unmyelinated primary afferent fibers called nociceptors, which terminate mainly in the superficial dorsal horn of the spinal cord. While most nociceptive fibers terminate in the spinal segment of the entrance, (collateral) fibers may ascend and descend several segments upon their entry into the spinal cord, which is reflected in the receptive fields of central nociceptive neurons. In chronic pain states like inflammatory or neuropathic pain, the area of nociceptive activity may expand even further in rostrocaudal and mediolateral directions. Also, within minutes (inflammatory pain) or days (neuropathic pain), an increased sensitivity of peripheral and central nociceptive neurons will develop, which is referred to as sensitization. While anatomical, physiological, and psychophysical techniques have focused on one particular aspect of central sensitization at a time, functional imaging techniques like functional MRI, intrinsic optical imaging, and autofluorescent flavoprotein imaging (AFI) are able to capture both spatial and temporal dimensions of central sensitization simultaneously. AFI and other neuroimaging techniques may clarify fundamental aspects relating to the spread of nociceptive activity within the spinal cord and may thus provide a practical tool to test the efficacy of new analgesic drugs or procedures in animals and ultimately in humans.

Correction for Cavanaugh et al., Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli

Correction for Cavanaugh et al., Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli

Localization of soluble guanylyl cyclase in the superficial dorsal horn

Nitric oxide (NO) has been implicated in pain processing at the spinal level, but the mechanisms mediating its effects remain unclear. In the present work, we studied the organization of the major downstream effector of NO, soluble guanylyl cyclase (sGC), in the superficial dorsal horn of rat. Almost all neurokinin 1 (NK1) receptor-positive neurons in lamina I (a major source of ascending projections) were strongly immunopositive for sGC. Many local circuit neurons in laminae I-II also stained for sGC, but less intensely. Numerous fibers, presumably of unmyelinated primary afferent (C fiber) origin, stained for calcitonin gene-related peptide or isolectin B4, but none of these was immunopositive for sGC. These data, along with immunoelectron microscopy results, imply that unmyelinated primary afferent fibers terminating in the superficial dorsal horn lack sGC. Double labeling showed that neuronal nitric oxide synthase (nNOS) seldom colocalized with sGC, but nNOS-positive structures were frequently closely apposed to sGC-positive structures, suggesting that in the superficial dorsal horn NO acts mainly in a paracrine manner. Our data suggest that the NK1 receptor-positive projection neurons in lamina I are a major target of NO released in superficial dorsal horn. NO may also influence local circuit neurons, but it does not act on unmyelinated primary afferent terminals via sGC.

A Specific Inhibitory Pathway between Substantia Gelatinosa Neurons Receiving Direct C-Fiber Input

The spinal substantia gelatinosa (SG) is a major termination region for unmyelinated (C) primary afferent fibers; however, how the input it receives from these sensory fibers is processed by SG neurons remains primarily a matter of conjecture. To gain insight on connections and functional interactions between intrinsic SG neurons, simultaneous tight-seal, whole-cell recordings were made from pairs of neurons in rat spinal cord slices to examine whether impulses in one cell generated synaptic activity in the other. Most SG neuron pairs sampled lacked synaptic interaction. Those showing a linkage included a recurring pattern consisting of a monosynaptic, bicuculline-sensitive inhibitory connection from an islet cell to a transient central neuron, each of which received direct excitatory input from different afferent C-fibers. This newly defined inhibitory circuit is postulated to represent a SG neural module by which a nociceptive C-fiber input to transient central cells is modified by other C-fiber messages.

Central projection of unmyelinated (C) primary afferent fibers from gastrocnemius muscle in the guinea pig.

We have demonstrated the central projections of muscle C or group IV afferent fibers in the guinea pig by tracing arborizations in the spinal cord. C afferent fibers from the gastrocnemius muscle (GCM) were electrophysiologically identified by conduction velocity (less than 1 m/second). A single neuron in the lumbar 5 dorsal root ganglion (L5 DRG) was intracellularly labeled with Phaseolus vulgaris leucoagglutinin (PHA-L). After iontophoretic injection of PHA-L, we processed the lumbar cord and L5 DRG for PHA-L immunohistochemistry. Six muscle C afferent fibers from 40 animals were labeled, and whole trajectories were recovered. Labeled fibers were reconstructed by tracing of the arbor in serial parasagittal sections. The GCM C afferents projected rostrocaudally for two or three segments and ran at the surface of the dorsal funiculus, giving off collaterals into laminae I and II and sometimes into parts of lamina III. We determined, based on the branching pattern and form of the terminal plexus, that the branching of muscle C afferent fibers showed an intermediate pattern that fell morphologically between the terminal patterns of somatic and visceral afferents. The numbers and sizes of fiber swellings and terminal swellings were measured on all collateral branches. We found that the area of distribution of the terminal swellings of muscle C afferent fibers is larger than that of somatic terminals but that the density of terminal swellings in the terminal area was lower than that of the somatic terminals.

Expression of vesicular glutamate transporters, VGluT1 and VGluT2, in axon terminals of nociceptive primary afferent fibers in the superficial layers of the medullary and spinal dorsal horns of the rat.

We examined immunohistochemically whether the vesicular glutamate transporters (VGluTs), VGluT1 and VGluT2, might be expressed in synaptic terminals of nociceptive primary afferent fibers within laminae I and II of the medullary and spinal dorsal horns of the rat. VGluT1 immunoreactivity (IR) was intense in the inner part of lamina II but weak in lamina I and the outer part of lamina II. VGluT2-IR was most intense in lamina I and the outer part of lamina II. Expression of VGluTs in synaptic terminals was confirmed by dual immunofluorescence histochemistry for VGluTs and synaptophysin. Expression of VGluTs in axon terminals of primary afferent fibers terminating in laminae I and II was also confirmed immunohistochemically after unilateral dorsal rhizotomy. The dual immunofluorescence histochemistry indicated expression of VGluTs in substance P (SP)-containing axon terminals in lamina I and the outer part of lamina II. Electron microscopy confirmed the coexpression of VGluTs and SP in axon terminals within laminae I and II; VGluTs was associated with round synaptic vesicles at the asymmetric synapses. It was further observed that isolectin IB4, a marker for unmyelinated axons, often bound with VGluT2-immunopositive structures but rarely with VGluT1-immunopositive structures in lamina II. Thus, the results indicated in laminae I and II of the medullary and spinal dorsal horns that both VGluT1 and VGluT2 were expressed in axon terminals of primary afferent fibers, including SP-containing nociceptive fibers and that VGluT in unmyelinated primary afferent fibers terminating in lamina II was primarily VGluT2.

Tachykininergic neurotransmission in the central nervous system

Tachykinins are widely distributed in mammalian central nervous system and exert a variety of actions through individual specific receptors. Neurotransmitter functions of substance P (SP), a member of mammalian tachykinins, have been firmly established in the spinal cord; SP is highly concentrated in the superficial layers of the dorsal horn, is released upon electrical stimulation, produces a slow excitatory postsynaptic potential in second-order neurons and is inactivated by peptidases. Since SP is contained in unmyelinated primary afferent fibers, which mediate nociception, SP is thought to transmit nociceptive information and contribute to occurrence of pathological pain states such as inflammation and nerve injury. Based on these findings, great effort has been devoted to developing NK-1 tachykinin receptor antagonists as a potent antinociceptive drug, but up to the present such effective drugs are unavailable. Tachykinin receptor antagonists have been also attracting much attention as a novel therapeutic drug for anxiety and depression other than pain. The amygdala, a key brain structure associated with emotional responses, is thought to be a target of tachykinin receptor antagonists for exerting psychopharmacological actions. Indeed, tachykinins enhance inhibitory synaptic transmission in the basolateral complex of the amygdala. Further study of tachykininergic transmission in the central nervous systems will open novel fields for pharmacology and therapeutics in neuropsychiatric disorders.

Presynaptic kainate receptors in primary afferents to the superficial laminae of the rat spinal cord

Subunits of glutamate receptors participate in the regulation of sensory transmission at primary afferent synapses in the superficial laminae of dorsal horn (DH). We report here on the distribution of kainate receptors (GluR5/6/7) in these laminae by using light microscope (LM) and electron microscope (EM) immunocytochemistry. Standard (4%) paraformaldehyde fixation resulted in immunostaining for GluR5/6/7 in perikarya and fine processes in lamina II, especially its inner part (IIi). Preembedding EM revealed immunostaining of dendrites, perikarya, and occasional terminals, presumed to be from primary afferent fibers, at the center of glomerular arrangements. In rats perfused with 0.5% paraformaldehyde, LM showed a more punctate staining, mainly in the ventral part of lamina IIi and lamina III, than in material fixed with 4% paraformaldehyde. Approximately two-thirds of GluR5/6/7 puncta were also immunostained with synaptophysin, suggesting that in material fixed with 0.5% paraformaldehyde, a large fraction of these are synaptic terminals. Double immunostained puncta disappear 4 days after dorsal rhizotomy, suggesting that most of GluR5/6/7-immunopositive terminals are from primary afferent fibers. EM material fixed with 0.5% paraformaldehyde confirmed the expression of GluR5/6/7 in numerous synaptic endings with morphology of primary afferents. To determine the type of primary afferent terminals that express GluR5/6/7, two neuroanatomic tracers were injected in the sciatic nerves. The lectin from Bandeiraea simplicifolia (IB4) is selectively taken up by unmyelinated primary afferent fibers that terminate in the outer part of lamina II (IIo) and dorsal part of lamina IIi, whereas the B subunit of the cholera toxin (CTB) is selectively taken up by a broader class of primary afferents which, in superficial DH, terminate mainly in laminae I, ventral part of IIi, and III. Approximately 20% of GluR5/6/7-immunoreactive puncta colocalized with IB4, whereas approximately 40% of GluR5/6/7-immunoreactive puncta colocalized with CTB. The present study shows that (1) GluR5/6/7 does not have a clear and consistent spatial relation with postsynaptic sites, (2) a large number of primary afferents express GluR5/6/7, and (3) these are not limited to one functional class. Thus, modulation by glutamate of primary afferent terminals by means of kainate receptors in the superficial laminae of DH may predominantly involve presynaptic mechanisms. J. Comp. Neurol. 436:275–289, 2001. © 2001 Wiley-Liss, Inc.

Presynaptic kainate receptors in primary afferents to the superficial laminae of the rat spinal cord

Subunits of glutamate receptors participate in the regulation of sensory transmission at primary afferent synapses in the superficial laminae of dorsal horn (DH). We report here on the distribution of kainate receptors (GluR5/6/7) in these laminae by using light microscope (LM) and electron microscope (EM) immunocytochemistry. Standard (4%) paraformaldehyde fixation resulted in immunostaining for GluR5/6/7 in perikarya and fine processes in lamina II, especially its inner part (IIi). Preembedding EM revealed immunostaining of dendrites, perikarya, and occasional terminals, presumed to be from primary afferent fibers, at the center of glomerular arrangements. In rats perfused with 0.5% paraformaldehyde, LM showed a more punctate staining, mainly in the ventral part of lamina IIi and lamina III, than in material fixed with 4% paraformaldehyde. Approximately two-thirds of GluR5/6/7 puncta were also immunostained with synaptophysin, suggesting that in material fixed with 0.5% paraformaldehyde, a large fraction of these are synaptic terminals. Double immunostained puncta disappear 4 days after dorsal rhizotomy, suggesting that most of GluR5/6/7-immunopositive terminals are from primary afferent fibers. EM material fixed with 0.5% paraformaldehyde confirmed the expression of GluR5/6/7 in numerous synaptic endings with morphology of primary afferents. To determine the type of primary afferent terminals that express GluR5/6/7, two neuroanatomic tracers were injected in the sciatic nerves. The lectin from Bandeiraea simplicifolia (IB4) is selectively taken up by unmyelinated primary afferent fibers that terminate in the outer part of lamina II (IIo) and dorsal part of lamina IIi, whereas the B subunit of the cholera toxin (CTB) is selectively taken up by a broader class of primary afferents which, in superficial DH, terminate mainly in laminae I, ventral part of IIi, and III. Approximately 20% of GluR5/6/7-immunoreactive puncta colocalized with IB4, whereas approximately 40% of GluR5/6/7-immunoreactive puncta colocalized with CTB. The present study shows that (1) GluR5/6/7 does not have a clear and consistent spatial relation with postsynaptic sites, (2) a large number of primary afferents express GluR5/6/7, and (3) these are not limited to one functional class. Thus, modulation by glutamate of primary afferent terminals by means of kainate receptors in the superficial laminae of DH may predominantly involve presynaptic mechanisms.

Central projections of trigeminal unmyelinated primary afferent fibers in the Japanese macaque

Central projections of trigeminal unmyelinated primary afferent fibers in the Japanese macaque

Are there long-term changes in the basal or evoked Fos expression in the dorsal horn of the spinal cord of the mononeuropathic rat?

The long-term changes in Fos like-immunoreactivity (Fos-LI) in the dorsal horn of the spinal cord following various peripheral nerve lesions remain controversial. This study considers such an approach with chronic constriction injury rats (CCI: loose ligations of the sciatic nerve), at 2 weeks after the surgery, when changes in spontaneous and evoked behaviour were clearly described. All rats used for Fos studies displayed allodynia to mechanical stimulation (decrease of 32% of the vocalization threshold to paw pressure). In CCI rats, which displayed `spontaneous pain-related behaviour', the number of Fos-LI neurones, in the absence of any intentional stimulation, was very low and comparable with that observed in normal and sham-operated rats (<10 neurones/40 μm section). Thus, in this model, the expression of Fos protein is not a reliable index of spontaneous pain. Surprisingly, despite the fact that in this model numerous anatomical studies described a dramatic loss of large and unmyelinated primary afferent fibers, we were unable to detect changes in the number and distribution of Fos-LI evoked by various modalities of peripheral noxious stimulation (noxious thermal stimuli, noxious mechanical stimuli and carrageenin induced inflammation). For example, the stimulus-response curves for the number of Fos-LI neurones evoked by a series of heat stimuli (40, 45, 48, 52, 55°C) were almost superimposable for CCI, sham-operated and normal rats. In contrast, stroking of the nerve-injured paw induced a significant expression of Fos-LI in the superficial laminae (I–II) of the dorsal horn of CCI rats (19.5±3/sections, P=0.027) which was greater than that observed in sham-operated (6.5±3/sections) or in normal rats (3.5±2/section). These modifications may reflect mechanical allodynia observed in behavioural studies and could be related to A β fibers, which are known to be severely affected after the constriction of the nerve. These results suggest that this approach could be useful to study, at the cellular level, in freely moving rats, some pharmacological aspects of neuropathic pain.

Fundamentals of pain perception in animals

Pain is mediated by functionally distinctive components. It may involve acute high threshold afferent stimuli (thermal, mechanical or chemically damaged tissue), protracted afferent input (long-lasting hyperalgesia) or low-threshold input (allodynia as related to pain from light touch). Behavioral patterns will be associated with the effects of noxious stimulus on excitatory transmitters. Humane studies using mechanical, thermal (cold pressor test), audio-evoked potentials, or other noxious stimuli during anesthesia and analgesia provide clues to perception of pain in animals and help us determine guidelines for the clinical relief of animal pain. There is a better understanding of cutaneous somatosensory responses than for deep sensation (e.g., subcutaneous tissue, muscle, bone, viscera). The prevention or treatment of pain can best be accomplished when there is a diagnosis of the neuroanatomical distribution of the pain based on evidence of sensory dysfunction involving a peripheral nerve, plexus, nerve root or central pathway using different modalities for quantitative sensory testing. Anatomical studies have demonstrated that unmyelinated primary afferent fibers contain a variety of neuroactive substances which may be released by high-intensity peripheral stimulation. Fast depolarization of the dorsal horn nociresponsive neurons is mediated by tissue damage. These neuroreceptors may be activated by glutamate, substance P, and neurokinin A. The major ascending pathway relaying nociceptive information relating to pain is the spinothalamic tract. Most of the neurons in this tract are nociceptive or thermoreceptive. The thalamus is the final relay nucleus for all the sensory systems, except olfactory tissue. Injury or disease processes in deeper tissue or visceral areas with extensive innervation may result in pain thresholds not adequately controlled by current available analgesics or at their recommended dosage levels. These issues are complex and diagnosis is even more difficult due to species and breed differences in outward expression to painful insult. Medications can now be targeted for specific receptors.

Sprouting of primary afferent fibers after spinal cord transection in the rat

After spinal cord injury, hyper-reflexia can lead to episodic hypertension, muscle spasticity and urinary bladder dyssynergia. This condition may be caused by primary afferent fiber sprouting providing new input to partially denervated spinal interneurons, autonomic neurons and motor neurons. However, conflicting reports concerning afferent neurite sprouting after cord injury do not provide adequate information to associate sprouting with hyper-reflexia. Therefore, we studied the effect of mid-thoracic spinal cord transection on central projections of sensory neurons, quantified by area measurements. The area of myelinated afferent arbors, immunolabeled by cholera toxin B, was greater in laminae I–V in lumbar, but not thoracic cord, by one week after cord transection. Changes in small sensory neurons and their unmyelinated fibers, immunolabeled for calcitonin gene-related peptide, were assessed in the cord and in dorsal root ganglia. The area of calcitonin gene-related peptide-immunoreactive fibers in laminae III–V increased in all cord segments at two weeks after cord transection, but not at one week. Numbers of sensory neurons immunoreactive for calcitonin gene-related peptide were unchanged, suggesting that the increased area of immunoreactivity reflected sprouting rather than peptide up-regulation. Immunoreactive fibers in the lateral horn increased only above the lesion and in lumbar segments at two weeks after cord transection. They were not continuous with dorsal horn fibers, suggesting that they were not primary afferent fibers. Using the fluorescent tracer DiI to label afferent fibers, an increase in area could be seen in Clarke's nucleus caudal to the injury two weeks after transection. In conclusion, site- and time-dependent sprouting of myelinated and unmyelinated primary afferent fibers, and possibly interneurons, occurred after spinal cord transection. Afferent fiber sprouting did not reach autonomic or motor neurons directly, but may cause hyper-reflexia by increasing inputs to interneurons.

Is there a nociceptive carousel?

Modern understanding of nociceptive mechanisms in the spinal cord has been gained in most part from two important discoveries: (1) co-release of excitatory amino acids and tachykinins [substance P (SP) and neurokinin A] from fine, unmyelinated primary afferent fibres, C-fibres, in the spinal dorsal horn and (2) NMDA (N-methyl-d-aspartate) receptor activation as the fundamental element of hyperexcitability of dorsal horn wide dynamic range (WDR) neurones. Tachykinins enhance responses of these cells to excitatory amino acids[1]; however, the precise interaction between these two neurotransmitter systems in spinal neuronal circuitry has never been fully understood.

Central projections of primary afferent fibers from the rat trigeminal nerve labeled with isolectin B4-HRP

The central projections of unmyelinated primary afferent fibers from the rat trigeminal nerve were investigated using retrograde and transganglionic transport of isolectin V4-horseradish peroxidase (B4-HRP). After the injection of a small amount of the tracer into various areas of the facial dermis and oral mucosa, small neuronal somata were retrogradely labeled in the trigeminal ganglion. Clearly delineated transganglionic labeling was observed exclusively in lamina II of the medullary and upper cervical dorsal horn. Its distribution showed a somatotopic organization. Fibers from the oral mucosa terminated in the region 0–1.7 mm caudal to the obex, whereas those from the facial skin terminated in the region from 1.3 mm caudal to the obex through the second cervical segment. In each of these two terminal regions, the rostral areas in the periphery were represented more rostrally than the caudal areas, and the dorsal areas in the periphery were represented more ventrolaterally than the ventral areas.

Central projections of primary afferent fibers from the rat trigeminal nerve labeled with isolectin B4-HRP

The central projections of unmyelinated primary afferent fibers from the rat trigeminal nerve were investigated using retrograde and transganglionic transport of isolectin V4-horseradish peroxidase (B4-HRP). After the injection of a small amount of the tracer into various areas of the facial dermis and oral mucosa, small neuronal somata were retrogradely labeled in the trigeminal ganglion. Clearly delineated transganglionic labeling was observed exclusively in lamina II of the medullary and upper cervical dorsal horn. Its distribution showed a somatotopic organization. Fibers from the oral mucosa terminated in the region 0–1.7 mm caudal to the obex, whereas those from the facial skin terminated in the region from 1.3 mm caudal to the obex through the second cervical segment. In each of these two terminal regions, the rostral areas in the periphery were represented more rostrally than the caudal areas, and the dorsal areas in the periphery were represented more ventrolaterally than the ventral areas.

1928 Central projections of unmyelinated primary afferent fibers from the trigeminal nerve: a study using isolectin b4-hrp in the rat

The I-B4 isolectin from Bandeiraea simplicifolia exhibits specific binding to a subpopulation of rat dorsal root ganglion neurons of small diameter which terminate in the substantia gelatinosa of the dorsal horn. Recent double-labelling experiments in the rat have demonstrated that only primary afferents which innervate the skin are recognized by the I-B4 lectin [Plenderleith and Snow (1993) Neurosci. Lett. (in press)]. As the I-B4 lectin appears to bind selectively to a subset of small-diameter primary afferents with cutaneous peripheral projections, we sought to determine whether it could be used as a transganglionic tracer which selectively labels the spinal terminations of cutaneous afferents in superficial dorsal horn. We now report that the I-B4-horseradish peroxidase conjugate labels synaptic terminals in lamina II of the dorsal horn following the injection of the conjugate into the sciatic and saphenous nerves in the rat. Electron-microscopic examination of the dorsal horn revealed many examples of labelled synaptic terminals and unmyelinated axons, but in no cases was label observed in myelinated axons. No label was observed outside of the substantia gelatinosa; thus the I-B4 isolectin is unique among lectins used for transganglionic tracing in that it does not retrogradely label motoneurons.These results, together with previous studies of lectin binding properties of primary sensory afferents, suggest that injection of I-B4 conjugates into peripheral nerves enables the visualization of the central terminations of cutaneous C-fibres. Transganglionic labelling with the I-B4 isolectin from Bandeiraea simplicifolia should facilitate further examination of synaptic relationships of nociceptive cutaneous afferents in the superficial dorsal horn.