Morphology and somatotopic organization of the central terminals of hindlimb hair follicle afferents in the rat lumbar spinal cord

The morphology of the central collateral arborizations of 24 A-beta hair follicle afferents (HFAs) innervating different regions of the skin of the hindlimb were studied by the intra-axonal injection of horseradish peroxidase (HRP) in adult rats. A total of 236 collaterals were recovered. These fell into three classes--complex, simple, and blind-ending--based on numbers of boutons and terminal branch patterns. The morphology of the HFA central arbors innervating the lateral and medial leg and dorsum of the foot was flame-shaped. Afferents with receptive fields on the glabrous-hairy skin border consistently had extra terminal branches running ventromedially into laminae IV/V. Differences in the width of terminal arbors were found. HFA terminals innervating the lateral leg formed narrower sheets than those innervating the dorsum of the foot and toes. The somatotopic organization of the collaterals and terminal arborizations of individual afferents were analyzed both by considering all the collaterals along an axon's rostrocaudal extent and by only examining arbors with boutons (the complex and simple arbors). Thirty-seven percent of blind-ending and 18% of simple collaterals were found to overlap in the rostrocaudal direction with the complex arborizations of afferents whose receptive fields were in a different cutaneous nerve territory. There was no overlap between complex arborizations of afferents from different nerve territories. However, the complex arbors of afferents with receptive fields within a particular nerve territory showed considerable terminal overlap even if they had nonadjacent peripheral receptive fields. The topographical organization of the central terminals of HFAs, forms a coarse somatotopic map of overlapping terminals whereby a particular region of dorsal horn has a maximal, but not exclusive, input from a particular area of skin.     

The differential contribution of capsaicin-sensitive afferents to behavioral and cardiovascular measures of brief and persistent nociception and to Fos expression in the formalin test

Intraplantar injection of dilute formalin evokes brief (Phase 1) and persistent (Phase 2) increases in primary afferent activity, pain behavior, and cardiovascular responses, and induces spinal cord Fos-like immunoreactivity (Fos-LI). Although previous studies demonstrated that the destruction of small diameter primary afferents with neonatal capsaicin treatment decrease formalin-evoked nociception, these studies only evaluated behavioral responses, and did not distinguish between Phase 1 and 2. To address these questions, we simultaneously evaluated formalin-evoked pain behavior (flinching of the afflicted paw), cardiovascular responses (heart rate and mean arterial pressure), and lumbar spinal cord Fos expression in control rats and in rats treated with capsaicin (100 mg/kg) one day postpartum. We found that neonatal capsaicin-treated rats, compared to controls, exhibited similar cardiovascular responses and slightly less flinching behavior during Phase 1. During Phase 2, however, capsaicin-treated rats exhibited 59% less flinching and 45% smaller heart rate responses. Also, in capsaicin-treated rats, we counted 59% fewer Fos-labeled neurons in the spinal cord. These results indicate that capsaicin-sensitive afferents contribute to formalin-evoked behavioral and cardiovascular responses and to spinal cord neuronal responses. The differential effect of neonatal capsaicin on nociception during Phase 1 and Phase 2 suggests that sensitization mechanisms during Phase 1 do not contribute to the magnitude of nociceptive responses during Phase 2.     

Neonatal Sciatic Nerve Section Results in a Rearrangement of the Central Terminals of Saphenous and Axotomized Sciatic Nerve Afferents in the Dorsal Horn of the Spinal Cord of the Adult Rat

Previous studies have shown that following neonatal peripheral nerve injury, adjacent intact myelinated and unmyelinated primary afferents sprout into the central denervated terminal area. The present study investigates this in more detail and goes further, to study the fate of the central terminals of the surviving axotomized primary afferent neurons. Bulk labelling of the sciatic and saphenous nerves with horseradish peroxidase conjugated to choleragenoid (B-HRP), to label the A fibres, or wheatgerm agglutinin (WGA-HRP), to label C fibres were employed to investigate the central consequences of sciatic nerve section and ligation on the day of birth, in adult rats. Bulk labelling of the axotomized sciatic or intact saphenous nerve with either tracer and comparison with contralateral controls revealed alterations to the terminal field. The intact saphenous nerve terminal field expanded caudally from mid L4 to the L4-L5 boundary when labelled with WGA-HRP and to the sacral cord when labelled with B-HRP. Labelling the axotomized sciatic nerve with either tracer revealed little change in the overall somatotopic organization of central terminals, although labelling was less intense compared to control nerves and more variable with WGA-HRP. Invasion of the substantia gelatinosa (SG) by axotomized A fibres was observed in segments L3-5, into the area occupied by axotomized C fibres. This area was also invaded by intact saphenous A fibres in the L4–5 segments. These results demonstrate that following neonatal nerve section: (i) axotomized primary afferents are able to retain a ‘normal’ somatotopic map in the rostrocaudal plane; (ii) both A and C fibres from adjacent intact nerves sprout into the denervated territory, but A fibres sprout further caudally; (iii) axotomized A fibres and invading intact A fibres both sprout dorsally into denervated SG. As a result, there is considerable overlap between nerve territories in denervated spinal cord, suggesting that competition for laminar termination sites exists between A and C fibres and also between axotomized and intact primary afferents.     

Neonatal capsaicin treatment prevents the normal postnatal withdrawal of A fibres from lamina II without affecting fos responses to innocuous peripheral stimulation

The development of spinal cord sensory pathways has been investigated in postnatal day (P) 21 rat pups following neonatal capsaicin treatment. Capsaicin-induced destruction of C fibres was confirmed by 62% loss of Isolectin B4 (IB4)-binding and an 86% loss of calcitonin gene-related peptide (CGRP)-immunoreactive small diameter dorsal root ganglion cells. Neonatal capsaicin treatment prevented the normal withdrawal of choleragenoid-horseradish peroxidase (B-HRP)-labelled A fibres from lamina II (substantia gelatinosa) to deeper laminae postnatally. A fibre terminals projected more dorsally, extending into 43% of lamina II compared to vehicle-treated littermates. A small cell loss in, and/or shrinkage of, substantia gelatinosa cannot account for this. These support the concept of a competitive interaction between A and C fibre afferents to establish final terminal fields. However the continued exuberant A fibre termination in capsaicin-treated rats did not lead to continued c-fos induction in the superficial dorsal horn by innocuous stimulation. In normal development, exuberant A fibre terminals coincide with c-fos activation in lamina II by innocuous skin stimulation [23]. Despite the continued presence of exuberant A fibre terminals, c-fos was not induced by innocuous peripheral stimulation in P21 capsaicin-treated rats implying that these superficial terminals do not activate lamina II neurons in the same way as in the neonate.     

Cutaneous hyperalgesia: contributions of the peripheral and central nervous systems to the increase in pain sensitivity after injury

This study assesses the contributions of the peripheral and central nervous systems in the development of hyperalgesia (increased pain sensitivity) after an injury. Experiments were carried out to examine the role of C-fiber afferents, the spinal cord and sympathetic efferents on inflammation, primary hyperalgesia and referred hyperalgesia produced in rats by a heat injury. A peripheral mechanism was indicated since both primary hyperalgesia and inflammation after a heat injury were significantly attenuated by blocking C-fiber afferents with local capsaicin. In addition, a central mechanism was indicated since the spread of hyperalgesia to the paw contralateral to a heat injury was prevented by either spinal anesthesia or the blocking of sympathetic efferents by guanethidine. A further role for central mechanisms was indicated since referred hyperalgesia--the enhancement of self-mutilation (autotomy) of a denervated limb which had previously sustained a heat injury--was reduced by spinal anesthesia or a combined blocking of C-fiber afferents and sympathetic efferents with intrathecal capsaicin + guanethidine. The results strongly suggest that referred hyperalgesia after a heat injury is dependent on increased spinal cord activity. However, autotomy in rats that did not undergo a previous injury was unaffected by either spinal anesthesia or intrathecal capsaicin. This suggests that spinal cord hyperactivity, although it plays a role in hyperalgesia following a heat injury, is not a crucial factor in producing pain and hyperalgesia after a nerve injury.     

Central terminations of cutaneous mechanoreceptive afferents in the rat lumbar spinal cord

The morphology in the dorsal horn of the lumbar spinal cord of the collateral branches and terminal arborizations of three different types of low-threshold mechanoreceptor innervating the skin of the rat hindlimb has been studied by the intracellular injection of horseradish peroxidase into physiologically characterized afferent fibres. The central terminals of five rapidly adapting glabrous skin mechanoreceptors (RA), six hair follicle afferents (HFA), and four slowly adapting type I afferent fibres (SA I; two from glabrous and two from hairy skin) were recovered for detailed analysis. The number of collaterals per axon varied from eight to 12, and the length of axon stained from 3.5 to 4.7 mm. In each afferent, while the majority (52-67%) of the terminal arborizations displayed extensive branching with large numbers of en passant and terminal synaptic boutons, the arborizations at the caudal and rostral extremes of the terminal field tended to be much simpler and less profuse, with few and in some cases no boutons. This did not appear to be the consequence of inadequate filling. The general pattern of the terminal arborizations was one of mediolaterally compressed, rostrocaudally oriented sheets. In the case of the RA afferents there was no overlap between the adjacent terminal arborizations; a few of the arborizations of the SA I afferents overlapped; most, but not all, of the HFA terminal arborizations overlapped. The terminal arborizations of the HFAs had a distinctive morphology identical to the flame-shaped arbors described in earlier Golgi studies and included synaptic boutons extending from inner lamina II to lamina IV. The morphologies of the RA and SA terminal arborizations were similar to each other, but the former tended to be concentrated in lamina IV with branches in III and V, whereas the latter tended to V with few branches more superficial than lamina IV. There were also differences in the intercollateral spacing between these two types of afferent fibre. While there are similarities between the morphology of the central terminals of cutaneous low-threshold mechanoreceptors in the rat and those previously described in the cat (for example, the longitudinally continuous arrangement of the mediolaterally restricted flame-shaped HFA arborizations and the discontinuous RA arborizations arising from a dorsally located axon), there are also some major differences: the large number of HFA arbors extending to lamina IIi and to lamina IV rather than being restricted to lamina III, the deeper location of the RA arbors (in laminae IV and V rather than lamina III),(ABSTRACT TRUNCATED AT 400 WORDS)     

The termination of primary afferents within the rat dorsal horn: Evidence for rearrangement following capsaicin treatment

The mode of termination of primary afferent fibres within the superficial dorsal horn of the spinal cord was studied with anterograde tracing methods in normal rats and in animals which had been treated with capsaicin (50 mg/kg. s.c.) shortly after birth. In normal animals following injections of horseradish peroxidase (HRP) into lumbar dorsal root ganglia L4, L5, L6, a Golgi-like filling of primary afferent fibres was seen consistently within laminae I, II, and III of the dorsal horn. Several types of lamina related arborisations were observed, depending upon the survival time after HRP injection. At the earliest survival time (1–2 days) a punctate granular labelling was found in laminae I and II inner (II_i). At 3 days survival a Golgi like labelling of primary afferent axons occurred and three tiers of arborisation were seen within laminae I, II outer (II_O), and II inner (II_i), respectively. By 7 days after injection this pattern was considerably reduced and there was coarser fibre labelling within laminae I, II_O, and III. When these tracing experiments were repeated in capsaicin-treated rats in which up to 90%of unmyelinated fibres had been destroyed, evidence for a considerable loss of some inputs and rearrangement of the remaining fibres was found. At 3–5 days survival the axonal labelling within laminae I and II_i was severely depleted yet some input, particularly to lamina II_O, was present. At 7 days survival deep fibre labelling within lamina III now extended well into lamina II. Mapping of the primary afferent input following ³H-proline injections into the dorsal root ganglion and subsequent autoradiographic processing confirmed that primary afferents terminated throughout the dorsal horn but most heavily in two bands within laminae I and II_i. These two dense layers of termination were not present in capsaicin-treated animals, suggesting that they corresponded to areas of unmyelinated primary afferent fibre input. These data, taken together with histochemical observations on the distribution of substance P and fluoride-resistant acid phosphatase (FRAP)-containing primary afferents, suggests that the earliest-labelled fibre systems (3days) were unmyelinated and replaced at 7 days by predominantly A6 myelinated fibres. It is further suggested that of the three tiers of C fibre terminations seen at 3 days, the most superficial corresponds in part to those primary afferents that can be stained for substance P within lamina I while the most ventral tier within lamina II_i are those primary afferents containing FRAP. The presence of the two temporally distinct fibre systems also suggests that laminae I and II_o, receive both C and A6 primary afferent input while lamina II_i, receives only C fibre input.     

Structure of the intraspinal projections of single, identified muscle spindle afferents from neck muscles of the cat

The morphology and frequency of collaterals originating from single afferents supplying primary endings of muscle spindles in dorsal neck muscles have been examined using intra-axonal injections of HRP. Within the segment in which the afferent entered the spinal cord, one collateral was found for every 3.3 mm of stained axon. In contrast, afferents--one of more segments rostral to the segment in which they entered the spinal cord--had fewer collaterals: One collateral was found for every 6.3 mm of stained axon. The branching structure and terminal distribution of the collaterals were generally similar regardless of the muscle from which the afferent originated and the segment in which the collateral was found. Boutons were found in 2 zones: One of these was located in the intermediate zone, within and around the central cervical nucleus, and the other was found in laminae VIII and IX, including the motoneuron nuclei. The ventral termination zone of collaterals in the same segment as their parent axon entered the spinal cord was larger and had more boutons than the same projection of collaterals whose parent axon entered the spinal cord 1 or 2 segments caudal to the segment in which the collateral was found. These results indicate that afferents supplying primary endings of neck muscle spindles are more likely to contact neurons in the same segment in which the afferent enters the spinal cord than in more rostral segments. However, even within the same segment in which the afferent enters the spinal cord, the projection of neck muscle afferents to the ventral horn is less dense than the corresponding projection of hindlimb muscle spindle afferents in the lumbosacral spinal cord.     

The effects of capsaicin applied to peripheral nerves on responses of a group of lamina I cells in adult rats

In adult rats, the sciatic and saphenous nerves on one side were treated topically with capsaicin. The capsaicin treatment had the effect of increasing the latency for withdrawal of the foot from hot water; 11–22 days later, the animals were decerebrated, and cells in the superficial dorsal horn of the lumbar cord with axons projecting in the contralateral dorsolateral funiculus (DLF) were examined electrophysiologically on the treated and untreated sides of the cord. HRP was applied to cut axons of the DLF at C4, in other rats, and retrograde labelling of cells in the lumbar cord indicated that most or all of the recordings in the capsaicin-treated animals were likely to originate from lamina 1. The dorsal horn cells, with receptive fields on the foot, showed decreased responses to electrically evoked afferent impulses in C fibres and grossly altered receptive fields. After capsaicin treatment, the proportion of cells responding to C afferents fell from 83% to 14%. The proportion responding only to C afferents and not to A afferents fell from 9% to 0%. The receptive fields (RFs) of these cells showed two gross abnormalities; 32% of the cells on the treated side had no apparent RF or an ill-defined, intermittent RF, whereas such cells were rare on the untreated side or in intact animals. By contrast 49% of the cells had grossly expanded RFs with an average area of 430 mm² against the normal average size of 130 mm². The antidromic conduction velocity of the axons of the cells was measured, and while there was no significant change in the number of cells with axons conducting above 3 m/second, an increased number of cells with lower velocities were recorded. It is suggested that the capsaicin treatment induced dendritic changes in cells with small axons allowing antidromic spikes to invade these cells.     

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.     

Fedotozine, a kappa-opioid agonist, prevents spinal and supra-spinal Fos expression induced by a noxious visceral stimulus in the rat.

Fedotozine, a kappa opioid agonist, reverses digestive ileus caused by acetic acid (AA)-induced visceral pain in rats. The aims of this study were: to map, in conscious rats, central pathways activated by AA using Fos as a marker of neuronal activation; to characterize primary afferent fibres involved in this activation; and to investigate the effect of fedotozine on AA-induced Fos expression. AA (0.6%; 10 mL kg-1) was injected i.p. in conscious rats either untreated; pretreated 14 days before with capsaicin; pretreated 20 min previously with fedotozine; or pretreated 2 h prior to fedotozine with the kappa-antagonist nor-binaltorphimine (nor-BNI). Controls received the vehicle alone. 60 min after injection of AA, rats were processed for Fos immunohistochemistry. Visceral pain was assessed by counting abdominal cramps. AA induced Fos in the thoraco-lumbar spinal cord (laminae I, V, VII and X) and numerous brain structures such as the nucleus tractus solitarius, and paraventricular nucleus (PVN) of the hypothalamus, whereas almost no Fos labelling was observed in controls. Capsaicin pretreatment blocked AA-induced Fos in all structures tested. Fedotozine significantly decreased AA-induced abdominal cramps and Fos immunoreactivity in the spinal cord and PVN, this effect being reversed by nor-BNI pretreatment. AA induces Fos in the spinal cord and numerous brain nucuei, some of which are involved in the control of digestive motility in rats. This effect is mediated through capsaicin-sensitive afferent fibres and prevented by fedotozine most likely through a peripheral action on visceral afferents.     

Plasticity of the serotonergic innervation of the dorsal horn of the rat spinal cord following neonatal capsaicin treatment.

Neonatal capsaicin treatments (25 or 50 mg/kg, 12, 24, or 48 hr after birth given subcutaneously) were applied in order to follow by immunocytochemical techniques the postnatal development and plasticity of the serotonergic system in the dorsal horn of the rat spinal cord. Two markers of the lesions of C primary afferents induced by capsaicin were tested by immunocytochemical detection: substance P and calcitonin gene-related peptide (CGRP). We show that the internal part of substantia gelatinosa (lamina Ili) which does not contain serotonergic fibers in intact or vehicle-treated rats is invaded within a few days after capsaicin treatment by serotonergic fibers apparently sprouting from the deepest laminae. Moreover, these fibers often establish axodendritic synapses while synapses are rare in intact animals in the whole dorsal horn. This reorganization is stable whatever the dose of capsaicin used or the moment chosen for its injection. On the other hand, while lesions of substance P-ergic fibers appeared quite stable, partial recovery of CGRP innervation was found after 3 to 6 months, especially with the low dose of capsaicin. We discuss the ability of the serotonergic system innervating the dorsal cord either to find new targets or to fill vacated sites when one of its putative targets is removed.     

Contribution of capsaicin-sensitive primary afferents to mechanical hyperalgesia induced by ventral root transection in rats: the possible role of BDNF

Objective: A recent study showed that brain-derived neurotrophic factor (BDNF) may play a role in the development of the neuropathic pain resulting from injury to motor efferent fibres, such as that in the ventral root transection (VRT) model. Capsaicin stimulation of afferent fibres was also shown to result in the release of BDNF into the spinal cord. Here, the effects of ablation of capsaicin-sensitive primary afferents (CSPAs) by local application of capsaicin on the sciatic nerve on VRT-induced mechanical hyperalgesia were observed.

Methods: The paw withdrawal mechanical threshold (PWMT) was measured before and then 1 and 3 days and 1, 2, 3, 4 and 6 weeks after VRT.

Results: The results showed that local application of capsaicin significantly inhibited the decrease in the PWMT induced by VRT, suggesting the inhibitory effect of locally delivered capsaicin. Furthermore, intrathecal administration of exogenous BDNF not only produced mechanical hyperalgesia but also significantly blocked the inhibitory effect of capsaicin.

Conclusion: Taken together, the results of this study suggest that CSPA fibres may contribute to mechanical hyperalgesia in the VRT model.     

Collateral sprouting of the central terminals of cutaneous primary afferent neurons in the rat spinal cord: pattern, morphology, and influence of targets

The capacity of the central terminals of primary afferents to sprout into denervated areas of neonatal spinal cord and the morphology of any novel terminals has been investigated. In rats which had undergone sciatic nerve section on the day of birth, 12 of 18 physiologically characterized intact saphenous hair follicle afferents (HFAs) were labelled intra-axonally with horseradish peroxidase (HRP) were shown to sprout up to 2,000 microns into the deafferented sciatic terminal field. The morphology of these sprouts depended on which area of the sciatic nerve territory was invaded by the afferent sprouts. Six HFAs sprouted into areas normally innervated by glabrous skin afferents and the morphology of the collateral sprouts in this region resembled that of rapidly adapting (RA) afferents. The other six saphenous HFAs had sprouted into sciatic "hairy" skin areas and the morphology of these sprouts, although abnormal, was flame shaped. In rats whose sural, saphenous, and superficial peroneal nerves were cut at birth, 4 of 7 single HRP labelled RA afferents had central terminals that had sprouted into regions of cord normally devoted to "hairy" input. These showed clear signs of HFA morphology despite their peripheral receptive fields remaining in the glabrous skin. The results show collateral sprouting of single cutaneous sensory afferent axons into adjacent inappropriate central target regions following neonatal deafferentation. Such plasticity may provide some compensation following neonatal injury. The morphology of the sprouted terminals is appropriate to the new target area rather than to its functional class and is also independent of the peripheral receptive field location providing an example of central rather than peripheral control over afferent growth patterns.     

Distribution of sural nerve afferent fibres within the dorsal horn of adult rats treated at birth with capsaicin

The distribution of sural nerve afferent fibres within the spinal cord of normal adult rats and of adult rats treated at birth with capsaicin was examined using transganglionic transport of horseradish peroxidase (HRP). Labelled fibres were seen, in normal and in capsaicin-treated rats, in Laminae I-VI of the central third of the dorsal horn, extending rostrocaudally between the L3 and L5 segments. It is concluded that the changes in dorsal horn somatosensory systems induced by neonatal capsaicin are not due to anatomical redistribution of the areas of termination of peripheral nerves within the spinal cord.     

Neurophysiological effects of capsaicin

Data obtained from neonatally treated rats are fairly consistent. However, there is disagreement as to whether mechanical and thermal nociceptive thresholds are elevated or unchanged in this group. There are at least two major areas of disagreement in adult animal capsaicin research. Behavioral data are extremely variable. The thermal nociceptive threshold after systemic capsaicin has been reported to be both raised and lowered. After intrathecal capsaicin injection, the thermal nociceptive threshold was reported raised, but onset and duration of responses varied and some animals exhibited no changes. Capsaicin application to peripheral nerve, however, drastically increased thermal threshold. Mechanical pain threshold has been reported both increased and unchanged after systemic capsaicin treatment and unchanged after intrathecal injection. Obviously, capsaicin's effects upon pain perception are not fully understood. Although lower on the phylogenetic scale than many mammals, rodents exhibit complex individualistic behavior. Lower vertebrates may eventually provide more simple behavioral models for pain tolerance.Investigators also disagree as to whether C fibres can conduct action potentials after local capsaicin application. C fibre conduction was reported unaffected by capsaicin in an acute preparation and for 13–21 days after treatment. On the other hand, C fibre compound action potentials have been reported diminished for up to 2 h after capsaicin application. Additional conduction impairment studies will be useful in comparing peripheral and intrathecal capsaicin application.There is general agreement that, allowing for variation in dosages and route of administration, capsaicin causes central and peripheral C fibre damage, though never as extensive in adults as in neonates. Neonatal capsaicin treatment (always s.c.) results in destruction of C and some Aδ fibres and their central terminals. Capsaicin causes degeneration of C terminals in the adult CNS only when applied centrally.In both neonates and adults, s.c. capsaicin depletes the putative ‘pain’ peptide neurotransmitter, SP, from peripheral and sensory neurons and the tissues they innervate but not from the gut. Capsaicin-induced SP depletion in neonates is permanent. Systemic administration to adult depleted SP from much the same areas as observed in neonates, but all areas but the medulla exhibited a slow, regional recovery.Intraventricular injection of capsaicin depleted SP in the adult medulla only, while other SP-containing areas affected by systemic injection remained intact. Intranigral capsaicin injection had no effect on SP content but depleted 5-HT and its metabolite, 5-HIAA, and dopamine metabolites. Direct application of capsaicin to the sciatic nerve may block axonal peptide transport in sensory C fibres but not in motor fibres.In neonates, capsaicin depleted peptide markers of primary afferents, i.e., SRIF, VIP, CCK, FRAP, GABA and opiate receptors from the spinal cord and/or DRG. Systemic administration to adults depleted SRIF and FRAP in the dorsal spinal cord but not opiate receptors or glutamic acid decarboxylase (GAD) activity. Probably because of capsaicin-induced structural and biochemical alterations, spinal, medullary, and peripheral neurons become at least temporarily less responsive, although not uniformly so.Neonatal capsaicin treatment abolished the C fibre mediated neurogenic oedema response to injury or irritation. Subcutaneous administration or direct (but not intrathecal) application of capsaicin to peripheral nerve temporarily blocked neurogenic oedema in adults. On a gross scale, pharmacological tests employing capsaicin are aimed at identifying the receptors it affects to explain complex visceral reflex mechanisms. Neurophysiological tests are more specific because so far, capsaicin has been found to affect only nociceptive primary afferents. At the cellular level, capsaicin depolarizes sensory terminals and exerts a Ca²⁺ dependent SP release in the spinal cord. Capsaicin prolongs sensory neuron action potentials, whereas opiates and opioid peptides shorten AP duration and inhibit SP release from spinal sensory terminals. Capsaicin's hyperpolarization of DRG neurons provides an interesting contrast that indicates differences in its effects on ganglionic, axonal, terminal and receptor sites. Also due to its specific action, capsaicin has been used to indicate presynaptic opiate receptors on SP-releasing spinal nociceptive terminals.     

Human and Rat Primary C-Fibre Afferents Store and Release Secretoneurin, a Novel Neuropeptide

Secretoneurin is a recently discovered neuropeptide derived from secretogranin II (SgII). Since this peptide could be detected in the dorsal horn of the spinal cord we studied whether it is localized in and released from primary afferent neurons. Secretoneurin was investigated with immunocytochemistry and radioimmunoassay in spinal cord, dorsal root ganglia and peripheral organs. SgII mRNA was determined in dorsal root ganglia. Normal rats and rats pre-treated neonatally with capsaicin to destroy selectively polymodal nociceptive (C-) fibres were used. Slices of dorsal spinal cord were perfused in vitro for release experiments. Immunocytochemistry showed a distinct distribution of secretoneurin-immunoreactivity (IR) in the spinal cord and lower brainstem. A particularly high density of fibres was found in lamina I and outer lamina II of the caudal trigeminal nucleus and of the spinal cord. This distribution was qualitatively identical in rat and human post-mortem tissue. Numerous small diameter and some large dorsal root ganglia neurons were found to contain SgII mRNA. Capsaicin treatment led to a marked depletion of secretoneurin-IR in the substantia gelatinosa, but not in other immunopositive areas of the spinal cord and to a substantial loss of small (<25 μm) SgII-mRNA-containing dorsal root ganglia neurons. Radioimmunoassay revealed a significant decrease of secretoneurin-IR in the dorsal spinal cord, the trachea, heart and urinary bladder of capsaicin-treated rats. Perfusion of spinal cord slices with capsaicin as well as with 60 mM potassium led to a release of secretoneurin-IR. In conclusion, secretoneurin is a neuropeptide which is stored in and released from capsaicin-sensitive, primary afferent (C-fibre) neurons. It may, therefore, be a novel peptidergic modulator of pain transmission or of C-fibre mediated non-nociceptive information.     

Dose-dependent effects of capsaicin on primary sensory neurons in the neonatal rat

The numbers of myelinated and unmyelinated fibers were counted in dorsal roots of adult rats treated neonatally with capsaicin in doses ranging from 5 to 100 mg/kg. Substance P and somatostatin levels in the spinal cord, dorsal roots, and sensory ganglia also were determined in control and treated animals. Capsaicin administration lead to the loss of both small myelinated and unmyelinated fibers from dorsal roots. However, whereas a near total loss, up to 94%, of unmyelinated fibers was achieved after high doses of capsaicin, the reduction of myelinated fibers, even of the smallest caliber, did not exceed 40%. The degree of fiber loss showed a clear dose dependency, with little detectable damage to myelinated fibers at doses of less than 50 mg/kg and with an ED50 for damage to unmyelinated fibers of 5 to 10 mg/kg. In all of the structures examined, particularly the dorsal roots, a roughly parallel decrease of substance P and somatostatin was found with capsaicin dose. The depletions of spinal cord substance P (55%) and somatostatin (20%) produced by neonatal capsaicin treatment were similar to those produced by dorsal rhizotomy. Capsaicin does not appear to be specific for primary afferents containing either substance P or somatostatin.     

Roles of capsaicin-sensitive primary afferents in differential rat models of inflammatory pain: a systematic comparative study in conscious rats

To characterize the role of capsaicin-sensitive primary afferents in inflammatory pain, the effects of subcutaneous (s.c.) injection of 0.15% capsaicin on different chemical irritants-induced pathological nociception including persistent spontaneous nociception, primary thermal and mechanical hyperalgesia, and inflammatory response were systematically investigated in unanesthetized conscious rats. Four different animal models of inflammatory pain: the bee venom (BV) test, the formalin test, the carrageenan model, and the complete Freund's adjuvant (CFA) model, were employed and compared. Local pre-treatment with capsaicin produced a significant inhibition on the s.c. BV and formalin induced long-lasting persistent spontaneous nociception. However, this capsaicin-induced inhibitory effect on spontaneous nociception in the BV test was only found within the late phase (tonic nociception; 11-60 min), but not the early phase (acute nociception; 0-10 min). A complete preventing effect of capsaicin on the decreased thermal paw withdrawal latency was found in the BV, carrageenan, and CFA models. Nevertheless, pre-treatment with capsaicin only produced complete blocking effects on the decreased mechanical paw withdrawal threshold in the BV and carrageenan models, but not in the CFA model. For inflammatory response, a significant inhibition of the BV-elicited paw swelling was found following capsaicin treatment. In marked contrast, capsaicin did not produce any effects on the paw inflammation during exposure to carrageenan, CFA, and formalin. These data suggest that capsaicin-sensitive primary afferents may play differential roles in the induction and development of pathological nociception in differential inflammatory pain models. In contrast to other chemical irritants, BV-induced long-term spontaneous nociception, facilitated nociceptive behavior, and inflammation are modulated by peripheral capsaicin-sensitive afferents.     

Paraventricular nucleus of the hypothalamus: an electrophysiological investigation of neurons projecting directly to intermediolateral nucleus in the cat

Most type I synaptic glomeruli were lacking in adult rats given capsaicin neonatally, while topical cord applications of the drug caused dense degeneration of numerous C_I- and very few C_IIa-terminals. These data give support to the presumable origin of C_I-terminals from unmyelinated primary afferents and suggest that C_IIa-terminals may belong to small myelinated afferents.