Mapping immunoreactive epitopes in the human peripheral nervous system using human monoclonal anti-GM1 ganglioside antibodies

A series of monoclonal IgM anti-GM1 ganglioside antibodies has been cloned from peripheral blood lymphocytes of patients with multifocal motor neuropathy and Guillain-Barré syndrome. In solid-phase immunoassay, the antibodies react with GMI, and also in differing degrees to the structurally related glycolipids asialo-GM1 (GA1) and GD1b. Here we describe the binding patterns of six human anti-GM I antibodies to epitopes within the human nervous system. Antibodies were observed to bind to motor neurons and spinal grey matter, dorsal and ventral spinal roots, dorsal root ganglion neurons, nodes of Ranvier, neuromuscular junctions and skeletal muscle. The distribution of immunoreactive epitopes, which included sensory structures, extended beyond those sites conventionally regarded as pathologically affected in anti-GM1 antibody-associated motor nerve syndromes. This undermines a model of disease pathogenesis based solely on antigen distribution. Factors other than the presence or absence of antigen, such as the local ganglioside topography, antibody penetration into, and pathophysiological vulnerability of a particular site may also influence the clinicopathological outcome of anti-GM1 antibody-mediated autoimmune attack.     

Clinical factors relating to the presence of serum anti-GM1 and GD1b antibodies in demyelinating neuropathy--study using a multivariate analysis

We studied clinical factors relating to the presence of serum anti-GM1 and GD1b antibodies in patients with demyelinating neuropathy using a multivariate analysis. Sera were obtained from 46 patients with chronic inflammatory demyelinating polyneuropathy (CIDP) and 33 with Guillain-Barré syndrome (GBS) and kept frozen at -20 degrees C until use. Anti-GM1 and GD1b IgM and IgG antibodies were measured by ELISA at serum dilution of 1:100 and considered to be positive when those values were more than the cut off values determined by means and standard deviations in 35 normal controls. Age, sex, duration, prodromal disease, neurological findings, concentration of CSF protein, nerve conduction, treatment, and outcome were investigated in all patients retrospectively. Multivariate logistic models using all those characteristics were used to clarify the clinical factors relating to the presence of anti-GM1 and GD1b antibodies. In CIDP, anti-GM1 antibodies associated with or without anti-GD1b antibodies were frequently seen in patients with motor dominant neuropathy than those with sensory dominant neuropathy (P = 0.007, odds ratio = 11.6). There was significant difference in anti-GM1 IgM antibodies (P = 0.003, odds ratio = 22.2), but no difference in IgG antibodies. Anti-GM1 antibodies were observed 5 (IgM, 5; IgG, 2) of 7 patients with pure motor neuropathy, 9 (IgM, 8; IgG, 4) of 17 with motor dominant neuropathy, 5 (IgM, 2; IgG, 3) of 16 with sensori-motor neuropathy, and none of 6 with sensory dominant neuropathy.(ABSTRACT TRUNCATED AT 250 WORDS)     

A case of biliobiliary fistula diagnosed by percutaneous transhepatic cholangioscopy

Autoimmune ataxic neuropathies are a subset of the sensory ataxic neuropathies which are characterized by ataxia as the dominant presenting feature. The major known causes of autoimmune ataxic neuropathies include sensory variants of the Guillain-Barré syndrome, including Miller-Fisher syndrome, subsets of immunoglobulin M paraproteinaemic neuropathy, paraneoplastic neuropathy and the neuropathy associated with Sj?gren's syndrome. Identified antigens as targets for autoantibodies include gangliosides, myelin associated glycoprotein, Hu antigen and extractable nuclear antigens. Some recent studies support the pathogenic role of anti-GD1b ganglioside antibody in autoimmune ataxic neuropathies. The major site of pathology in autoimmune ataxic neuropathies is the dorsal root ganglion, but dorsal roots and peripheral nerve myelin and axons may also be affected.     

Anti-GM1 ganglioside antibodies cloned from autoimmune neuropathy patients show diverse binding patterns in the rodent nervous system

We have recently cloned a panel os monoclonal IgM anti-GM1 ganglioside antibodies from peripheral blood lymphocytes of patients with multifocal motor neuropathy and Guillain Barré syndrome. In solid-phase immunoassay, the antibodies all reacted with GM1 and also reacted to different degrees with the structurally related glycolipids asialo-GM1 and GD1b. These antibodies are being used to study the pathogenesis of the anti-GM1 antibody-medicated neuropathy in different experimental systems. In the present immunofluorescence study we report the binding patterns of 5 of these antibodies in the rodent nervous system. The antibodies demonstrated highly diverse binding patterns on tissue sections and teased fibers when compared to one another and between species. The antibodies bound many central and peripheral nervous system structures, including neurons and myelin, motor end plate regions, and muscle spindles. The diversity of binding shown by these antibodies provide evidence that may account for the differing clinical phenotypes, including normality, associated with elevated titers of anti-GM1 antibodies.     

Sulfated glucuronyl glycolipids and gangliosides in the optic nerve of humans

Pathologically delayed visual evoked potentials may be present in patients with neuropathy associated with IgM M-proteinemia, which is directed against myelin-associated glycoprotein and sulfated glucuronyl glycolipids (SGGLs), but there are no reports of these antigens in the optic nerve. We recently examined human optic nerve and occipital lobe tissues for the occurrence of SGGLs using the technique of immunostaining on thin-layer chromatographic plates and found them in the optic nerve, but not the occipital lobe. SGGLs in the optic nerve may represent target antigens for CNS involvement by the M-protein in patients with neuropathy. We also studied the ganglioside composition of the optic nerve and found it different from that of the brain. Human optic nerve is characterized by an abundance of the b-series gangliosides, including GD1b, GT1b, and GQ1b. GD1a, which is usually a major component of brain gangliosides, is only a minor species of the optic nerve ganglioside fraction.     

Cholinergic neuron-specific ganglioside GQ1b alpha a possible target molecule for serum IgM antibodies in some patients with sensory ataxia

In neurological diseases the presence of certain anti-glycosphingolipid antibody species is associated with the clinical features. We recently isolated the novel cholinergic neuron-specific gangliosides GQ1b alpha and GT1a alpha from bovine brain. A monoclonal antibody specific for GQ1b alpha and GT1a alpha reacted strongly with the dorsal born of human spinal cord but not with human motor neurons. We investigated the serum antibodies to these minor gangliosides in a number of neurologic diseases and found that 4 patients with sensory ataxic neuropathy had a remarkably high IgM anti-GQ1b alpha antibody titer. GQ1b alpha may be a target molecule for serum IgM antibodies in some patients with sensory ataxic neuropathy.     

The sword of Damocles: the psychosocial impact of familial spinocerebellar ataxia in South Africa

It has been recently recognized that increased titers of serum anti-GM1 antibodies may be associated with motoneurone diseases or with multiple motor neuropathy with or without conduction block and also with chronic sensorimotor neuropathy and Guillain-Barré syndrome. Santoro et al. were the first to note that anti-GM1 antibodies were able to bind to the nodes of Ranvier of the sural nerve of a patient with clinical signs and symptoms mostly resembling amyotrophic lateral sclerosis who also showed, in nerve conduction studies, multifocal motor nerve fibers conduction block and serum IGM anti-GM1 antibodies. The two patients presented in this report had asymetrical motor neurone disease with signs and symptoms of lower motoneurone involvement, and other signs, in the first patient, which suggested the existence of upper motoneurone damage. Besides, the second patient also had clinical sensory impairment in the lower limbs. Electrophysiologically, none of them had nerve conduction block but both showed inexcitable median and sural nerve sensory fibers. Both had high titers of anti-GM1. A sural biopsy of both patients showed immunoglobulins into the sensory fibers. However, we do not know whether the anti-GM1 antibodies bind to a cross-reactive glycolipid other than the GM1 itself. In any case, it seems that the presence of anti-GM1 antibodies might be a marker signalling a potentially treatable immune disorder which may have signs of lower and upper motor neurone disease and, also, clinical and electrophysiological evidences of peripheral sensory involvement.     

Acute and chronic neuropathies: new aspects of Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy, an overview and an update

During the last 15 years new information about clinical, electrophysiological, immunological and histopathological features of acute and chronic inflammatory neuropathies have emerged. Thus, the Guillain-Barré syndrome (GBS) is no longer considered a simple entity. Subtypes of the disorder besides the typical predominant motor manifestation, are recognized, i.e. a cranial nerve variant with ophthalmoplegia, ataxia and areflexia, an immune-mediated primary motor axonal neuropathy (AMAN), and a motor-sensory syndrome (AMSAN). Also, the clinical pattern of GBS is related to preceding viral or bacterial infections. Two types of acute motor paralysis have been described, one with slow and incomplete recovery, another with recovery times identical with acute inflammatory demyelinating polyneuropathy (AIDP). Histologically, the first is characterized by Wallerian degeneration of motor roots and peripheral motor nerve fibres. In the latter anti-GM antibodies bind to the nodes of Ranvier producing a failure of impulse transmission. Motor-point biopsies have shown denervated neuromuscular junctions and a reduced number of intramuscular nerve fibres. Molecular mimicry has been postulated as a possible mechanism triggering GBS. Thus, in the cranial variant antibodies to ganglioside GQ1b recognizes similar epitopes on Campylobacter jejuni strains and similar observations apply to anti-GM1 antibodies. Chronic inflammatory demyelinating polyneuropathy (CIDP) also has several different clinical presentations such as a pure motor syndrome, a sensory ataxic variant, a mononeuritis multiplex pattern, relapsing GBS, and a paraparetic subtype. Each of the acute and the subtypes have different, more or less distinct, electrophysiologic and pathological findings. Instructive patient stories are presented together with there electrophysiologic and biopsy findings.     

Does hysteroscopy improve upon the sensitivity of dilatation and curettage in the diagnosis of endometrial hyperplasia or carcinoma?

We report the occurrence of a relapsing, severe predominantly motor neuropathy in a 75-year-old man with an IGM-K M-protein binding to gangliosides GM2, GM3, GM4, GD1a, GT1b and LM1. Motor nerve conduction velocities were slowed with conduction block. A superficial peroneal nerve biopsy specimen revealed segmental demyelination and remyelination. The patient improved after repeated plasma exchanges, and the antibody titer decreased in association with clinical recovery. This IgM M-protein has a unique, previously unreported binding specificity for terminal NeuAcalpha2-3Galbeta- moiety in common to all gangliosides bound by the antibody except GM2. M-proteins with this affinity may be involved in the pathogenesis of this and other cases of motor-dominant demyelinating neuropathy.     

Structural characterization of the disialogangliosides of murine peritoneal macrophages

Sialoglycosphingolipids (gangliosides) have been increasingly implicated as regulators of membrane signaling events. Macrophage ganglioside patterns dramatically increase in complexity when murine peritoneal macrophages are stimulated in vivo with the appearance of the sialidase-sensitive monosialoganglioside GM1b (cisGM1) as a major component. Gangliosides from stimulated murine peritoneal macrophages were separated into monosialo and polysialo fractions and the polysialo fraction structurally characterized by enzymatic, chemical, and mass spectra methods. All detectable components of the polysialo fraction were determined to be disialogangliosides. Treatment of the polysialo fraction with Clostridium perfringens sialidase produced mostly the sialidase-resistant monosialoganglioside, GM1a, and a minor amount of asialoGM1. Periodate oxidation and mass spectrometry analyses demonstrated the lack of tandem disialo moieties which indicated the absence of GD1b or GD1c (GD1) entities. The combined data showed the major disialogangliosides consisted of GD1a entities comprising IV3-NeuAc,II3NeuAc-GgOse4Cer, IV3-NeuGc,II3NeuAc-GgOse4Cer, IV3NeuAc,II3NeuGc-GgOse4Cer, and IV3-NeuGc,II3NeuGc-GgOse4Cer. Minor components consisted of GD1alpha entities, IV3NeuAc, III6NeuAcGgOse4Cer, IV3NeuGc, III6NeuGc-GgOse4Cer, and also positional isomer(s) of GD1alpha(NeuAc, NeuGc). These isomeric components were identified by collision analysis and tandem mass spectrometry. Consistent with previous analyses, the ceramide portion of all polysialo (disialo) gangliosides contained solely C18 sphingosine with C16 and C24 fatty acid moieties. These results, combined with the previous characterization of macrophage monosialogangliosides, indicate normal murine macrophage ganglioside biosynthesis proceeds along the "a" ganglioside pathway, e.g., GM3-->GM2-->GM1a-->GD1a, and the proposed asialoganglioside or "alpha" pathway, asialoGM1-->GM1b-->GD1alpha. The presence of totally sialidase-sensitive gangliosides appears to be characteristic of functional murine peritoneal macrophages while they are reduced in genetically impaired cells.     

Human dorsal root ganglion neurons from embryonic donors extend axons into the host rat spinal cord along laminin-rich peripheral surroundings of the dorsal root transitional zone

Following dorsal root crush, the lesioned axons regenerate in the peripheral compartment of the dorsal root, but stop at the boundary between the peripheral and the central nervous system, the dorsal root transitional zone. We have previously shown that fibres from human fetal dorsal root ganglia grafted to adult rat hosts are able to grow into the spinal cord, but were not able to specify the route taken by the ingrowing fibres. In this study we have challenged the dorsal root transitional zone astrocyte boundary with human dorsal root ganglion transplants from 5-8-week-old embryos. By tracing immunolabelled human fibres in serial sections, we found that fibres consistently grow around the dorsal root transitional zone astrocytes in laminin-rich peripheral surroundings, and extend into the host rat spinal cord along blood vessels, either into deep or superficial laminae of the dorsal horn, or into the dorsal funiculus. Human fibres that did not have access to blood vessels grew on the spinal cord surface. These findings indicate, that in spite of a substantial growth capacity by axons from human embryonic dorsal root ganglion cells as well as their tolerance to non-permissive factors in the mature mammalian CNS, these axons are still sensitive to the repellent effects of astrocytes of the mature dorsal root transitional zone. Furthermore, this axonal ingrowth is consistently associated with laminin-expressing structures until the axons reach the host spinal cord.     

BDNF attenuates functional and structural disorders in nerves of galactose-fed rats

Galactose intoxication of rats was used to disrupt metabolism of Schwann cells and skeletal muscle, two sites that contain the polyol-forming enzyme aldose reductase (AR). Galactose-fed rats develop a neuropathy characterized by nerve conduction deficits and axonal atrophy. To investigate the possibility that galactose metabolism by AR influences axonal function and structure by altering production of neurotrophic factors, the impact of galactose intoxication on nerve and muscle BDNF levels and the effects of exogenous BDNF treatment on galactose neuropathy were examined using biochemical, electrophysiologic and morphometric techniques. Galactose feeding increased BDNF protein in peripheral nerve and muscle. Exogenous BDNF treatment attenuated motor nerve conduction velocity deficits in the sciatic nerve of galactose-fed animals and myelin splitting of motor axons in the ventral root. In contrast, sensory nerve conduction velocity (SNCV) deficits in the sciatic nerve and myelin splitting in the central projections of sensory neurons were not prevented by BDNF treatment. BDNF treatment did not attenuate reduced axonal caliber in the sciatic nerve, but did ameliorate the diminution of the caliber of central sensory projections in the dorsal root. These findings point to the potential use of BDNF in the treatment of peripheral neuropathies.     

Mitochondria and cells produce reactive oxygen species in virtual anaerobiosis: relevance to ceramide-induced apoptosis

Antibodies reactive with the core glycan of asialoganglioside (GA1), monosialoganglioside (GM1), and disialoganglioside (GD1a) were studied in human sera. In healthy individuals, GA1-, GM1-, and GD1a-reactive antibodies were mainly of the IgM class, but also of the IgA and IgG classes, and were present at low titers in the serum of 68%, 79%, and 91% of the individuals studied, respectively. Levels of anti-GA1 and anti-GM1 antibodies, mainly of the IgA and IgG classes, were significantly elevated (P < 0.001) in 62% and 72% of subjects, respectively, chronically infected with Trypanosoma cruzi, with no association found with the degree of myocardial damage. No significant increase in anti-GA1 and anti-GM1 antibodies was found in dilated cardiomyopathy patients. The level of anti-GD1a antibody was not significantly different between healthy controls and chronic chagasic or dilatatory cardiomyopathy patients. Since the peripheral nervous system is very rich in gangliosides, it is possible that the increases in GA1- and GM1-specific antibodies that develop during chronic T. cruzi infection are involved in the pathology of peripheral neuropathy in Chagas' disease.     

Differential expression of gangliosides and galactolipids in fetal human oligodendrocytes and astrocytes in culture

The phenotypic expression of gangliosides and galactolipids was investigated using primary cultures of fetal human oligodendrocytes and astrocytes. These glial cells were isolated from fetal human brains of 12-18 weeks' gestation. Expression of gangliosides and galactolipids in oligodendrocytes and astrocytes was investigated by double labeling immunocytochemistry using rabbit antibodies specific for galactocerebroside (GalC, a cell type-specific marker for oligodendrocyte) and glial fibrillary acidic protein (GFAP, a cell type-specific marker for astrocyte) in combination with a panel of mouse monoclonal antibodies which react with specific gangliosides or galactolipids. A considerable number of GalC+ oligodendrocytes expressed intense immunoreactivities specific for GM3 (19%) and GM2 (45%) gangliosides. Approximately 11% of GalC+ oligodendrocytes expressed GM4 immunoreactivity, and smaller numbers of GalC+ oligodendrocytes expressed GD3 (4%), GD2 (1%), GT1b (5%) and A2B5 (3%) immunoreactivities. However, GalC+ oligodendrocytes did not express GM1, GD1a, GT1b or GQ1c. Major populations of GalC+ oligodendrocytes immunolabeled by rabbit anti-GalC antibody reacted with anti-GalC mAb (Ranscht mAb, 81%) or by anti-sulfatide mAb (O4 mAb, 91%). A considerable number of GFAP+ astrocytes expressed intense GM2 (26%) and GD2 (15%) immunoreactivities, while a smaller population expressed intense GM3 (3%), GD3 (6%) and GM4 (4%) immunoreactivities. Weak immunoreactions specific for GD1b, A2B5 and sulfatide were found in less than 1% each of GFAP+ astrocytes, while GFAP+ astrocytes did not express GM1, GD1a, GT1a, GT1b or GQ1b. These results indicate that GM3, GM2 and sulfatide are expressed in a major population of GalC+ oligodendrocytes, while GM3, GM2, GD3, GD2, and GM4 are expressed in a small but distinctive population of GFAP+ astrocytes. Our results suggest that GM4, GM1 and GD3, which are utilized as markers for adult human oligodendrocytes and myelin, are not the major ganglioside constituents in cultured fetal human oligodendrocytes.     

A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons after nerve injury

Several lines of evidence suggest that neurotrophin administration may be of some therapeutic benefit in the treatment of peripheral neuropathy. However, a third of sensory neurons do not express receptors for the neurotrophins. These neurons are of small diameter and can be identified by the binding of the lectin IB4 and the expression of the enzyme thiamine monophosphatase (TMP). Here we show that these neurons express the receptor components for glial-derived neurotrophic factor (GDNF) signaling (RET, GFRalpha-1, and GFRalpha-2). In lumbar dorsal root ganglia, virtually all IB4-labeled cells express RET mRNA, and the majority of these cells (79%) also express GFRalpha-1, GFRalpha-2, or GFRalpha-1 plus GFRalpha-2. GDNF, but not nerve growth factor (NGF), can prevent several axotomy-induced changes in these neurons, including the downregulation of IB4 binding, TMP activity, and somatostatin expression. GDNF also prevents the slowing of conduction velocity that normally occurs after axotomy in a population of small diameter DRG cells and the A-fiber sprouting into lamina II of the dorsal horn. GDNF therefore may be useful in the treatment of peripheral neuropathies and may protect peripheral neurons that are refractory to neurotrophin treatment.     

Miller Fisher anti-GQ1b antibodies: alpha-latrotoxin-like effects on motor end plates

In the Miller Fisher syndrome (MFS) variant of the Guillain-Barré syndrome, weakness is restricted to extraocular muscles and occasionally other craniobulbar muscles. Most MFS patients have serum antibodies against ganglioside type GQ1b of which the pathophysiological relevance is unclear. We examined the in vitro effects of MFS sera, MFS IgG, and a human monoclonal anti-GQ1b IgM antibody on mouse neuromuscular junctions (NMJs). It was found that anti-GQ1b antibodies bind at NMJs where they induce massive quantal release of acetylcholine from nerve terminals and eventually block neuromuscular transmission. This effect closely resembled the effect of the paralytic neurotoxin alpha-latrotoxin at the mouse NMJs, implying possible involvement of alpha-latrotoxin receptors or associated downstream pathways. By using complement-deficient sera, the effect of anti-GQ1b antibodies on NMJs was shown to be entirely dependent on activation of complement components. However, neither classical pathway activation nor the formation of membrane attack complex was required, indicating the effects could be due to involvement of the alternative pathway and intermediate complement cascade products. Our findings strongly suggest that anti-GQ1b antibodies in conjunction with activated complement components are the principal pathophysiological mediators of motor symptoms in MFS and that the NMJ is an important site of their action.     

The expression of P2X3 purinoreceptors in sensory neurons: effects of axotomy and glial-derived neurotrophic factor

We have studied the distribution and regulation of the P2X3 receptor (a ligand-gated ion channel activated by ATP) in adult dorsal root ganglion (DRG) neurons using a polyclonal antibody. P2X3 receptor immunoreactivity was normally present in about 35% of L4/5 DRG neurons, virtually all small in diameter. In the dorsal horn, P2X3 receptor expression was restricted to the terminals of sensory neurons terminating in lamina IIinner. P2X3 receptors were expressed in approximately equal numbers of sensory neurons projecting to skin and viscera but in very few of those innervating skeletal muscle. P2X3 receptors were found mostly in sensory neurons that bind the lectin IB4. After sciatic nerve axotomy, P2X3 receptor expression dropped by more than 50% in L4/5 DRG. Glial cell line-derived neurotrophic factor (GDNF), delivered intrathecally, completely reversed axotomy-induced down-regulation of the P2X3 receptor. We conclude that P2X3 receptors are normally expressed in nociceptive primary sensory neurons, predominantly the nonpeptidergic nociceptors. P2X3 receptors are down-regulated following peripheral nerve injury and their expression can be regulated by GDNF.     

Human immunoglobulin M paraproteins cross-reactive with Neisseria meningitidis group B polysaccharide and fetal brain

Three hundred fifty-nine serum samples from patients with immunoglobulin M (IgM) or IgG monoclonal gammopathies were tested for binding to the capsular polysaccharide (PS) of Neisseria meningitidis group B (MenB PS, poly-alpha[2-->8]-N-acetylneuraminic acid). Of 159 IgM paraproteins, 7 (4.4%) were positive, compared with 0 of 200 IgG paraproteins (P < 0.05). Since MenB PS reactivity was limited to the IgM paraproteins, the 159 IgM paraproteins were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with seven other bacterial PSs. None reacted with meningococcal A or C, Haemophilus influenzae type b, or Streptococcus pneumoniae type 3, 6, 14, or 23 PS. The specificity of the MenB PS-reactive antibodies was confirmed by demonstration of binding to N. meningitidis group B cells but not to a capsular PS-deficient mutant and by specific inhibition of binding to solid-phase MenB PS by soluble MenB PS in an ELISA. Five of five antibodies tested protected infant rats from bacteremia caused by Escherichia coli K1, an organism with a PS capsule that also is composed of poly-alpha[2-->8]-N-acetylneuraminic acid. Each of the seven MenB PS-reactive paraproteins had autoantibody activity as defined by binding to homogenates of calf brain in a radioimmunoassay. For six of the seven antibodies, binding to calf brain was inhibited by the addition of soluble MenB PS. Thus, approximately 4% of human IgM paraproteins have autoantibody activity to poly-alpha[2-->8]-N-acetylneuraminic acid, an antigen expressed in fetal brain and cross-reactive with the MenB capsular PS. The reason for this skewing of the IgM paraprotein repertoire toward reactivity with poly-alpha[2-->8]-N-acetylneuraminic acid antigenic determinants is unknown.     

Down-regulation of mu-opioid receptors in rat and monkey dorsal root ganglion neurons and spinal cord after peripheral axotomy

To understand the role of opioids and their receptors in chronic pain following peripheral nerve injury, we have studied the mu-opioid receptor in rat and monkey lumbar 4 and 5 dorsal root ganglion neurons and the superficial dorsal horn of the spinal cord under normal circumstances and after peripheral axotomy. Our results show that many small neurons in rat and monkey dorsal root ganglia, and some medium-sized and large neurons in rat dorsal root ganglia, express mu-opioid receptor-like immunoreactivity. Most of these neurons contain calcitonin gene-related peptide. The mu-opioid receptor was closely associated with the somatic plasmalemma of the dorsal root ganglion neurons. Both mu-opioid receptor-immunoreactive nerve fibers and cell bodies were observed in lamina II of the dorsal horn. The highest intensity of mu-opioid receptor-like immunoreactivity was observed in the deep part of lamina II. Most mu-opioid receptor-like immunoreactivity in the dorsal horn originated from spinal neurons. A few mu-opioid receptor-positive peripheral afferent terminals in the rat and monkey dorsal horn were calcitonin gene-related peptide-immunoreactive. In addition to pre- and post-junctional receptors in rat and monkey dorsal horn neurons, mu-opioid receptors were localized on the presynaptic membrane of some synapses of primary afferent terminals in the monkey dorsal horn. Peripheral axotomy caused a reduction in the number and intensity of mu-opioid receptor-positive neurons in the rat and monkey dorsal root ganglia, and of mu-opioid receptor-like immunoreactivity in the dorsal horn of the spinal cord. The decrease in mu-opioid receptor-like immunoreactivity was more pronounced in the monkey than in the rat dorsal root ganglia and spinal cord. It is probable that there was a parallel trans-synaptic down-regulation of mu-opioid-like immunoreactivity in local dorsal horn neurons of the monkey. These data suggest that one factor underlying the well known insensitivity of neuropathic pain to opioid analgesics could be due to a marked reduction in the number of mu-opioid receptors in the axotomized sensory neurons and in interneurons in the dorsal horn of the spinal cord.