Upregulation of the axonal growth and the expression of substance P and its NK1 receptor in human allergic contact dermatitis

Nerve fibers and sensory neuropeptides substance P and calcitonin gene-related peptide (CGRP) have been reported to be involved in allergic contact dermatitis (ACD). In the present study, we investigated the general innervation (using antibody against protein gene product 9.5, PGP 9.5), axonal growth (using antibody against growth associated protein, GAP-43), CGRP, and substance P with its receptor neurokinin 1 (NK1), in positive epicutaneous reactions to nickel sulphate from nickel-allergic patients, at the peak of inflammation, 72 hr after challenge with the antigen. There was an increased (p < 0.01) number of GAP-43 positive fibers in the eczematous compared with control skin, indicating an increased axonal growth already at 72 hr postchallenge. Double staining revealed a coexpression of CGRP and GAP-43 on dermal nerve fibers. There was no difference in the number of substance P and CGRP positive nerve fibers between eczematous and control skin. However, semiquantification analyses showed an increased expression of substance P positive inflammatory cells, being CD3, CD4, or CD8 positive, and NK1R positive inflammatory cells, being tryptase or CD3 positive. These results indicate a contribution of regenerating nerve fibers and substance P to the contact allergic reaction.     

Study of innervation, sensory neuropeptides, and serotonin in murine contact allergic skin

Density of nerve fibers, axonal growth, calcitonin gene-related peptide (CGRP), and substance P, and serotonin immunoreactivity as well as concentration were all determined in a murine model of contact allergy. Female Balb/c mice were sensitized on the back with oxazolone and 6 days later challenged with the same antigen on the dorsal surface of the ears, while control mice received the vehicle only. Then, 24 hr postchallenge, one ear was processed for immunohistochemical staining, while the other was frozen and processed for gas chromatography-mass spectrometry or radioimmunoassay (RIA). Protein gene product 9.5 (PGP 9.5) positive nerve fibers showed a tendency to increase in inflamed ears versus control ears in epidermis as well as the dermis. Growth-associated protein-43 (GAP-43) positive fibers in the epidermis were increased (p < .01) in inflamed ears, compared with control ears, as was the case for the dermal fibers, indicating increased axonal growth. Total (epidermis and dermis) numbers of CGRP and substance P positive nerve fibers tended to increase in the inflamed skin in contrast to control skin. In contrast, RIA demonstrated a lower (p < .05) concentration of CGRP in the inflamed ears compared with controls and a tendency for substance P to decrease in concentration in eczematous ears versus controls. There was no difference in serotonin concentration, or in the number of serotonin positive mast cells, between the inflamed and control skin, whereas semiquantification of serotonin positive platelets showed an increase in the inflamed (+/+) compared with control ears (+). Our results indicate that 24 hr after being challenged with the antigen, at the peak of murine skin inflammation, axonal growth, sensory neuropeptides, as well as serotonin may be involved.     

Expression of neuropeptides and growth-associated protein 43 (GAP-43) in cutaneous and mucosal nerve structures of the adult rat lower lip after mental nerve section.

The reinnervation of the adult rat lower lip has been investigated after unilateral section of the mental nerve. Rats were sacrificed at 4, 7, 9, 14, 30, and 90 days after the operation. A further group of animals with section of the mental nerve and block of the alveolar nerve regeneration, was sacrificed at 14 days. Specimens were processed for immunocytochemistry with antibodies against PGP 9.5, GAP-43 or neuropeptides (CGRP, SP and VIP). Four days after nerve section, axonal degeneration seems evident in the mental nerve branches and inside skin and mucosa. GAP-43 immunoreactivity is intense in the mental nerve 7 days after nerve section and it reaches its maximal expression and distribution in peripheral nerve fibres at 14 days. At 30 days, the decline in its expression is associated with the increase of PGP9.5-, SP-, and CGRP immunopositivity. VIP is observed only in perivascular fibres at all times observed. Present results suggest that, after sensory denervation of the rat lip, nerve fibres in skin and mucosa remain at lower density than normal. The different time courses in the expression of neuropeptides and GAP-43 suggest a possible early involvement of GAP-43 in peripheral nerve regeneration.     

Reciprocal age-related changes in GAP-43/B-50, substance P and calcitonin gene-related peptide (CGRP) expression in rat primary sensory neurones and their terminals in the dorsal horn of the spinal cord and subintima of the knee synovium

Age-related changes in the expression of the growth associated protein GAP-43/B-50, and the neuropeptides substance P and calcitonin gene-related peptide (CGRP) were investigated in the sensory neurones of rat dorsal root ganglia, dorsal horns of the spinal cord and subintimal knee synovium. The two time-points studied were 2 months (young adults) and 14-month (aged)-old Sprague Dawley rats. Dorsal root ganglia: In young adults, 40 and 35% of the L4-L5 dorsal root ganglion neurones were positive for GAP-43/B-50 with a 1.5 fold increase in frequency in aged rats at the L5 ganglion. GAP-43/B-50 was strongly expressed by the non-neuronal satellite cells of some medium and many large sized neurones in aged rats. There were marked reciprocal shifts between small and medium sized sensory neurones in respect to their substance P and CGRP expression profiles. Dorsal horn of the spinal cord: there was a 1.3 fold decrease of substance P at L5 level and a 1.3 and 1.5 fold decrease of CGRP at L4-L5 levels in aged rats, respectively. Synovial membrane: There was a 2.3 fold increase in GAP-43/B-50 and a 2.5 fold decrease of CGRP with no changes in substance P expression. These results indicate that (i) primary sensory neurones undergo age-related changes already in early stages of aging, (ii) aging may result in a reduction of substance P and CGRP axonal transport, and (iii) reduced numbers of CGRP containing synovial perivascular fibres may imply a deficient regulation of the synovial microvasculature and therefore metabolic homeostasis of the joint in aged subjects.     

Inflammation-induced increase in the density of neuropeptide-immunoreactive nerve endings in rat skeletal muscle

The density of substance P (SP)-, calcitonin gene-related peptide (CGRP)- and vasoactive intestinal polypeptide (VIP)-immunoreactive (ir) nerve endings was quantitatively evaluated in intact and inflamed gastrocnemius-soleus muscle of the rat. In persistently inflamed muscle (12 days after a single injection of Freund’s adjuvant into the muscle), the density of SP-ir fibres was significantly increased. CGRP- and VIP-ir fibres displayed an insignificant increase in density. The density of fibres ir for nerve growth factor (NGF) and for growth-associated protein 43 (GAP-43/B-50), a marker for axonal sprouting, regeneration and synaptic reorganisation, increased significantly in persistently inflamed muscle. The data are consistent with the established contribution of NGF on the expression of SP and GAP-43 in afferent neurones under the influence of a persistent inflammation. Received: 8 September 1997 / Accepted: 12 February 1998     

Neurovascular plasticity in the knee joint of an arthritic mouse model

Lower numbers of neuropeptide-containing fibers in arthritic joints have been found as compared to control joints. This may be the result of fiber depletion, necrosis of fibers, or proliferation of soft tissues without neural sprouting. To discriminate between these possibilities, we studied the relationships between soft tissue proliferation, changes in vascularity of synovial tissues, and changes in joint innervation during arthritis. Arthritis was induced in the knee joint of mice by a single subpatellar injection of methylated bovine serum albumin after previous immunization. Antibodies to protein gene product 9.5, S-100, and growth-associated protein-43 (GAP-43) were used to study the general innervation pattern. Antibodies to calcitonin gene-related peptide (CGRP), vasointestinal polypeptide (VIP), substance P (SP), and tyrosine hydroxylase (TH) were used to localize sensory (SP, CGRP, VIP) and sympathetic (TH) fibers. Blood vessels of the joint were studied with ink perfusion, GAP-43, and a vascular marker (LF1). Directly after the induction of arthritis, the synovial cavity was enlarged and filled with leukocytes. From day 4 onward, small sprouting blood vessels penetrated the avascular mass of cells in the joint cavity. After 1 week, the vascular sprouting activity and GAP-43 immunoreactivity were maximal, and after 2 weeks, vascular sprouting activity diminished. In the subsequent period, the synovia slowly regained their prearthritic appearance and thickness. The most pronounced changes in the general staining pattern of CGRP, SP, VIP, and TH were found in the periosteum. From 2 days to 4 weeks after the induction of arthritis, the layer of SP, CGRP, and VIP fibers in the femoral periosteum was thicker and more irregular. GAP-43 staining showed many terminal varicosities, which suggested sprouting of nerve fibers. From 2 days to 2 weeks after the induction of arthritis, the SP and CGRP fibers in the periosteum showed gradual depletion. In the thickened subsynovial tissues that were revascularized, no ingrowth of neural elements was found. As the total number of nerve fibers in the synovial tissue did not change, large parts of the synovia directly facing the joint cavity were not innervated at 1 week after the induction of arthritis. These results strongly suggest that periosteal SP and CGRP fibers were depleted during arthritis. Synovial proliferation without concomitant fiber growth is the main cause of the reduced number of immunocytochemically detectable fibers in the mouse arthritic knee joint.     

Distribution of GAP-43 nerve fibers in the skin of the adult human hand

Skin is an important region of somatic sensory input, and is one of the most innervated areas of the human body. In this study, we investigated in human hand skin the distribution of nervous structures immunoreactive for the growth-associated protein 43 (GAP-43) and the protein gene product 9.5 (PGP 9.5). GAP-43 is a neuronal presynaptic membrane protein that is generally considered to be a marker of neuronal plasticity. PGP 9.5 is a neuron-specific soluble protein that is widely used as general marker for the peripheral nervous system. The entire neural network of the dermis and epidermis was stained with antibody to PGP 9.5. In the dermis, there were fewer GAP-43-immunostained nerve fibers than PGP 9.5-immunostained nerve fibers, whereas in the epidermis the numbers were equal. Only some Merkel cells and Meissner corpuscles were GAP-43-immunoreactive. In conclusion, our results show that GAP-43 protein is expressed in a subset of PGP 9.5-immunoreactive nerve structures.     

大鼠自体移植脾组织GAP-43+神经再生的实验研究

目的 研究大鼠自体脾组织移植术后移植脾组织GAP-43^ 神经纤维再生及分布规律。方法 健康Wistar大鼠105只，随机分为自体脾组织移植组和对照组，于术后7、15、30、60、90、120、180d取移植脾组织标本，应用免疫组化方法显示GAP-43^ 神经纤维，并用图像分析系统，对不同时相点免疫组化GAP-43^ 染色区域进行图像定量分析。结果 自体脾组织移植术后30d，移植脾组织周围大网膜内可见GAP-43^ 神经纤维，并向移植脾组织内伸展；自体脾组织移植术后60～120d，移植脾组织内再生神经纤维逐渐增多；术后180d，移植脾组织中GAP-43^ 神经纤维分布及密度接近正常。结论GAP-43^ 神经纤维于脾移植术后60d开始出现，术后180dGAP-43^ 神经纤维接近正常脾。     

Distribution of nerve fibers during the development of palatine glands in rats

Background

Salivary gland maturation and function are modulated by the nervous system. Nevertheless, little is known about salivary gland innervation during development, particularly minor salivary glands. This study investigated the development of the innervation of the palatine glands of rat.

GAP-43 and p75NGFR immunoreactivity in presynaptic cells following neuromuscular blockade by botulinum toxin in rat

Summary Peripheral nerve lesion results in changes in protein expression by neurons and denervated Schwann cells. In the present study we have addressed the question whether similar changes take place following functional denervation. Using immunohistochemistry and immunoelectron microscopy we examined changes in growth-associated protein (GAP-43) and low-affinity nerve growth factor receptor (p75^NGFR) in rat gastrocnemius muscle following botulinum toxin-induced paralysis. GAP-43 and p75^NGFR were selected because they are not expressed by mature intact motor neurons or Schwann cells, but are expressed following nerve lesion in both motor neurons and denervated Schwann cells. In control muscle, GAP-43 and p75^NGFR immunoreactivity was seen only in nerve fibres near blood vessels. Two weeks after toxin injection, GAP-43 immunoreactivity could be seen at the motor endplates and in axons. Intensity of staining increased with longer survival and reached a peak between 4 and 8 weeks post-injection. Ultrastructurally, GAP-43 immunoreactivity was confined to nerve terminals and axons, whereas Schwann cells remained negative. Immunostaining for p75^NGFR also increased following toxin injection and was detected in some terminal Schwann cells and in perineurial cells of small nerve fascicles near the paralyzed target cells, but not in axons. These results show that changes in expression of GAP-43 in motor neurons following functional denervation closely resemble the changes following anatomical interruption of nerve-muscle contact. GAP-43 was not expressed in Schwann cells, indicating that its upregulation in these cells is induced by loss of axonal contact or nerve degeneration products. There is no support for a role of p75^NGFR in incorporation of neurotrophins in axons. The restriction of p75^NGFR expression to terminal Schwann cells and perineurial cells in close proximity to the paralyzed target suggests a role for a target-derived signal or, alternatively, macrophages in eliciting this expression.     

The tachykinin NK(1) receptor antagonist SR140333 prevents the increase of nerve growth factor in rat paw skin induced by substance P or neurogenic inflammation

Target-derived nerve growth factor provides trophic support for adult primary afferent neurons containing calcitonin gene-related peptide and tachykinins. Noxious chemical or thermal stimuli cause the release of these mediators from peripheral afferent nerve endings. However, little is known of the extent to which these mediators, in turn, influence nerve growth factor expression in the innervated tissue. The aim of this study was therefore to investigate the possible effect of exogenous substance P, or neurogenic inflammation on the nerve growth factor concentration in the skin of the rat hindpaw. Our results show that substance P as well as topical application of mustard oil cause a significant increase in detectable nerve growth factor, an effect that was prevented by treatment of rats with the tachykinin NK(1) receptor antagonist SR140333. We did not observe a significant inhibitory effect of SR140333 on the nerve growth factor content in non-treated skin, or the nerve growth factor increase caused by carrageenan or allergic inflammation.The results provide evidence that substance P as well as neurogenic inflammation cause a rapid increase in detectable nerve growth factor in the paw skin and suggest the involvement of NK(1) receptors in this effect. We obtained no evidence for the participation of a NK(1) receptor-mediated nerve growth factor increase in models of inflammation induced by non-neurogenic stimuli.     

Age-related changes and the possible adaptability of rat jaw muscle spindles: immunohistochemical and fine structural studies

Afferent signals from jaw muscle spindles contribute to the feedback mechanism that regulates mastication. The integrity and adaptability of this proprioceptor to age-related changes of the surrounding structures are therefore essential to maintain an appropriate masticatory function throughout life. In this study, we examined muscle spindles obtained from temporal and masseter muscles of 10-week-, 12-, 18-, and 24-month-old Wistar rats, employing immunohistochemistry for protein gene product 9.5 (PGP 9.5) or growth-associated protein (GAP-43) in addition to transmission electron microscopy, in order to investigate their morphological changes in relation to the effect of aging on the adaptive potential of the receptors. Immunohistochemistry for PGP 9.5 showed virtually similar reactions at sensory nerve terminals in all age groups. On the other hand, immunoreactivity for GAP-43 in the sensory nerve ending of the muscle spindles was found 2 and 3 weeks after birth but became almost undetectable by 10 weeks. However GAP-43 immunoreactions occasionally reappeared in those of spindles in 12- and 18-month old animals, and vanished again by 24 months of age. Electron microscopic observations also revealed age-related morphological changes in the intrafusal muscle fibers of the rats in 12-month and older groups. The extent of degenerative and/or atrophic alterations of intrafusal fibers increased with age and involved the nerve elements of spindles by 24 months. These findings indicate that the adaptation potential of rat jaw muscle spindles is well preserved until middle age, but diminishes in elderly animals. Structural changes of muscle spindles in elderly animals probably contribute to the deterioration of the muscular function.     

The Peridural Membrane of the Human Spine is Well Innervated

There is evidence that low back pain may originate from a peridural membrane (PDM) at the inferior and medial aspect of neural foramen of the lumbar spine. The objective of this investigation was to determine if this membrane contains neural elements suggestive of sensory innervation with nociceptive function. Spines of four embalmed and three non‐embalmed human cadavers were dissected using a sagittal approach to the neural foramen. Seventeen samples of the peridural membrane overlying the neural foramen were collected for immunohistochemistry (IHC) examination by light microscopy and transmission electron microscopy (TEM). Chromagin tagged antibodies to protein gene product 9.5 (PGP9.5) and S‐100, and fluorescent antibodies to substance P and calcitonin gene related peptide (CGRP) were used to label neural structures in tissue sections cut from paraffin embedded blocks. This approach allows good visualization of all neural elements, small sensory, and nociceptive nerve fibers in particular. Neural elements were found in all samples. Marked presence of small nerve fibers was observed in 12 of 15 samples. IHC and TEM evaluation revealed myelinated as well as unmyelinated fibers in the peridural membrane. CGRP and substance P immunoreactive fibers indicative of nociceptive function were abundant. These findings confirm and expand evidence that the peridural membrane in human is well innervated and contains sensory nociceptive nerve fibers suggestive of a nociceptive function of the membrane. Anat Rec, 299:484–491, 2016. © 2016 Wiley Periodicals, Inc.     

大鼠坐骨神经损伤与再生中GAP－43表达的实验研究

用免疫组化技术对６０只大鼠坐骨神经切断、卡压和再卡压损伤后不同时期的ＧＡＰ－４３表达作了观察．观察结果：（１）大鼠坐骨神经卡压或切断后，前角运动神经元、后根神经节细胞和坐骨神经纤维均产生免疫反应阳性，其中７ｄ组近段和１４ｄ组远段坐骨神经纤维为强阳性，（２）３０ｄ组各部免疫反应减弱，６０ｄ组基本恢复正常；（３）３０和６０ｄ组的再卡压损伤的各部均较同期其他损伤组免疫反应为强；（４）伤侧腰骶髓前角运动神经元染色逐渐减弱．ＧＡＰ－４３免疫反应结果显示，神经元胞体部分随再生期延长而逐渐减弱，周围部由近至远逐渐增强，又逐渐恢复至正常水平．结果提示ＧＡＰ－４３主要由神经元胞体所产生，随轴突逐渐转运至损伤和再生处，表明了此种蛋白参与神经再生，在神经再生过程中一直起着重要的作用．     

自体移植脾组织内GAP-43神经的生成机制

为探索自体移植脾组织内GAP-43神经的再生机制,将Wistar大鼠42只随机分为实验组和对照组。实验组切除脾脏以后,切取1／2脾脏,切成1 mm×1 mm×1 mm大小的组织块植入大网膜内,术后7、14、30、60、90、120、180 d取脾组织标本通过原位杂交检测GAP-43 mRNA、NGF mRNA和TrkA mRNA,同时进行免疫组化染色观察GAP-43神经。对照组手术松动脾脏,在术后相同时间点取脾组织作对照观察。结果显示:术后30 d检测到移植脾组织内有GAP-43 mRNA、NGF mRNA和TrkA mRNA表达,90 d达高峰后开始下降;术后60 d明显可见GAP~43染色阳性神经纤维,90 d密度最大,主要存在于血管周围,以后无明显改变。结果提示:自体移植脾组织内GAP-43神经再生与内源性NGF和TrkA表达密切相关。     

Cellular reactions to axotomy in rat superior cervical ganglia includes apoptotic cell death

The cellular response to axonal injury in the superior cervical ganglion was examined by immunofluoresence at intervals from 6 h to 14 days after transection of the internal and external carotid nerves. GAP-43-immunoreactivity (IR) appeared in some neurons in the ganglia 1 day after axotomy, while neurons in control ganglia were GAP-43 negative. In 3 days axotomized ganglia GAP-43-IR structures were increased in number and intensity in nerve fiber bundles, while GAP-43-positive perikarya were restricted to the middle and caudal parts of the ganglia and showed an intensity that was stronger than at 1 day after axotomy. These GAP-43-positive neurons were also galanin positive. In the cranial part of the ganglia, S100-IR in satellite cells was weak at 18 h after axotomy. Peripheral to this area, S100-IR was stronger and co-localized with HSP-72-IR, preferentially located in satellite cells. HSP-72-IR was, however, occasionally observed also in principal neurons at 1 and 3 days after axotomy. In eosin-stained sections, neurons and satellite cells in the cranial part of 1 day axotomized ganglia were reduced in number, and a further loss was noted at 3 days. At 12 h some satellite cells in the cranial part of the ganglia were labelled by the in situ DNA 3'-end labelling method, indicating apoptosis, and at 18 h many cells were labelled. Some neuronal perikarya were also labelled in this region. Labelling was not observed at 1 day or later after axotomy, nor in control ganglia. The results may imply that not only neurons but also satellite cells react to neuronal axonal injury with apoptosis. Neurons in the middle and caudal part of the ganglia survived and showed increased content of GAP-43 and galanin, possibly a sign of regeneration/neuronal plasticity.     

CGRP肽能神经在皮肤组织中的分布及其意义

目的:了解降钙素基因相关肽(caleitonin gene-related peptide,CORP)在皮肤组织中的分布、超微定位,以探讨其在皮肤中功能影响.方法:采用ABC-GND免疫组化染色、免疫透射电镜方法观察CGRP在正常皮肤分布和超微结构情况.结果:CGRP分布在表皮下、毛囊、皮脂腺周围、真皮、皮下以及小血管壁周围,其中以小动脉壁阳性纤维密度大.可见粗、细两种纤维,前者位于真皮深层及皮下,后者位于表皮下、毛囊皮脂腺周围.四肢较胸背皮肤CGRP分布相对多而明显.结论:CGRP可能作为神经调节或神经递质参与皮肤各种生理功能调节.尤其在血管周围分布,可能作为CGRP调节皮肤的机制研究和可能的药物治疗的研究基础.     

GAP-43免疫反应神经纤维对发育中大鼠脾脏支配的研究

本实验用免疫组化方法研究了含生长相关蛋白（GAP－43）的神经纤维在新生、幼年、成年大鼠脾脏的分布及发育规律、结果显示，在出生第1d，GAP－43免疫反应阳性神经纤维团簇即出现于尚在形成中的白髓（原基）中．根据形态可将它们分粗直和细网两种类型．至生后第10d，随着脾脏的迅速发育，GAP－43免疫反应阳性神经纤维的密度也达最高峰，它们大量出现于脾脏的血管系统周围和白、红髓内，其形态演变为具有膨体的细纤维．在生后20d，除脾脏的血管周围外，GAP－43阳性纤维密度有所下降，只出现于脾小体和被膜等结构内。在成年大鼠脾脏，除上述部位外，阳性纤维明显见于发育成熟的小梁中。本研究发现，GAP－43免疫反应阳性神经纤维密度在脾脏发育各阶段有所不同，以生后第10d为最多．本研究还就含GAP－43神经在发育中脾脏的分布规律与含TH能神经成分做了比较．     

Markers for vitiligo related neuropeptides in human skin nerve fibers

Skin distribution of substance P (SP)-, somatostatin (SOM)-, calcitonin-gene-related peptide (CGRP)- and neuropeptide-Y (NPY)-like immunoreactivity in vitiligo patients was studied by an indirect immunofluorescence technique. Immunocytochemical characteristics of the epidermis, dermoepidermal junction, papillary and reticular dermis, and skin appendages were analyzed in lesional and marginal vitiligo areas as well as in healthy skin. SP-, SOM-, CGRP-, and NPY-immunoreactive nerve fibers were observed in healthy pigmented skin, with patterns specific for immunoreactive distribution. Thin SP-containing fibers were observed in dermal papillae, extending into the epidermis, and SP-immunoreactive nerve fibers were seen around blood vessels and sweat glands. SOM-immunoreactive varicose nerve fibers were associated with Meissner's corpuscles in dermal papillae, while CGRP-like immunoreactivity was demonstrated in free subepidermal nerve terminals and sensory nerve fibers around blood vessels, hair follicles and sweat glands. Autonomic NPY-containing nerve fibers innervated eccrine sweat glands and blood vessels. The distribution of these neuropeptides was the same in healthy controls, except for an increased immunoreactivity to NPY and to a lesser extent to CGRP. These results suggest that NPY may serve as a neurochemical marker in the pathogenesis of the disease, thus supporting the neuronal theory of vitiligo.     

GAP-43 immunoreactivity is widespread in the autonomic neurons and sensory neurons of the rat.

GAP-43 is a membrane-bound phosphoprotein generally associated with axon growth during development and regeneration. Using immunohistochemical and immunoblotting techniques this study shows that GAP-43 is expressed extensively in the unperturbed adult autonomic nervous system. Strong immunoreactivity was seen in the developing and mature enteric subdivision of the autonomic nervous system and in nerves of the iris and various blood vessels. The presence of GAP-43 immunoreactivity in varicose nerve fibres, and a comparison of the labelling pattern of GAP-43 with the nerve associated marker PGP 9.5 suggests that GAP-43 is present in most or all autonomic nerve fibres in these organs. Immunoblotting of gut samples on 10% polyacrylamide gels revealed a single band of approximately 45,000 mol. wt that co-migrated with pure central nervous system GAP-43. Surgical sympathectomy experiments resulting in almost complete elimination of sympathetic fibres did not markedly affect the pattern of GAP-43 immunoreactivity in the iris, indicating that GAP-43 is expressed not only in sympathetic nerves but also in parasympathetic and sensory fibres. These findings show that GAP-43 is expressed extensively in autonomic nerves of the adult rat, at levels comparable to those seen during development. High levels of GAP-43 are not therefore restricted to development and regeneration in this part of the nervous system.