α-Bungarotoxin binding sites on sensory neurones and their axonal transport in sensory afferents

The autoradiographic localization of [¹²⁵I]α-bungarotoxin binding sites on primary sensory fibres was investigated. Nicotinic α-bungarotoxin binding sites were localized to a small sub-population of large dorsal root ganglion cells in the rat, monkey, cat and human dorsal root ganglia. Ligation of the sciatic nerve or dorsal root in the rat resulted in an anterograde accumulation of binding sites proximal to the dorsal root ganglion, and a small retrograde accumulation. Unilateral dorsal root section in the rat produced a loss of toxin binding sites mainly within lamina III of the dorsal horn. These results suggest that nicotinic α-bungarotoxin binding sites manufactured in large dorsal root ganglion cell bodies are transported both centrally to the spinal cord and also peripherally.     

Identification of a nicotinic acetylcholine receptor on neurons using an alpha-neurotoxin that blocks receptor function

An alpha-neurotoxin, Bgt 3.1, that reversibly blocks the ACh response of chick ciliary ganglion neurons has been used to identify 2 classes of high-affinity binding sites on the cells in culture. The first class appears to be the alpha-bungarotoxin binding site on the neurons. The second class of Bgt 3.1 sites is distinct from the alpha-bungarotoxin binding sites and has the properties expected for the functional nicotinic ACh receptor on the cells. Equilibrium binding and kinetic studies indicate a Kd value of 5-6 nM for Bgt 3.1 at the second class of sites. The kinetics and affinity of binding are consistent with those inferred from previous physiological studies for Bgt 3.1 inhibition of receptor function. Bgt 3.1 binding to the sites is completely inhibited by each of the cholinergic ligands ACh, carbachol, nicotine, d-tubocurarine, and trimethaphan, but not by alpha-bungarotoxin. Highest site densities are found in cultures of ciliary and sympathetic ganglion neurons, cell types known to have ganglionic nicotinic ACh receptors. Low levels of sites may be present in cultures of spinal cord and dorsal root ganglion neurons; no binding is found in cultures of skeletal myotubes or cardiac cells when alpha-bungarotoxin is used to block Bgt 3.1 binding to alpha-bungarotoxin sites. These results demonstrate that Bgt 3.1 can be used as a specific probe for the nicotinic ACh receptor on chick autonomic neurons.     

Neural nicotinic acetylcholine responses in sensory neurons from postnatal rat

The whole-cell configuration of the patch-clamp technique was used to study nicotinic acetylcholine (ACh) responses in freshly dissociated dorsal root ganglion (DRG) cells from postnatal rat. At negative holding potentials with physiological solutions in the bath and the pipette, ACh (20 microM), nicotine (5 microM) or DMPP (20 microM) activated inward currents in 51% of the cells. Average current density was higher in 1-month-old compared to newborn animals. Nicotinic agonist-induced currents were unaffected by atropine (10 microM) but reversibly blocked by hexamethonium (20 microM). Although labeling with fluorescent alpha-bungarotoxin (BGT) demonstrated the presence of toxin binding sites on DRG cells, DMPP-induced inward currents were unaffected by micromolar BGT. Neuronal bungarotoxin (100 nM), in contrast, led to a largely irreversible block of the nicotinic responses. These results show that postnatal DRG cells express functional nicotinic acetylcholine receptors (nAChR) of a neuronal type.     

Trigeminal and dorsal root ganglion neurons express CCK receptor binding sites in the rat, rabbit, and monkey: possible site of opiate-CCK analgesic interactions.

125I-Bolton-Hunter sulfated cholecystokinin-8 was used to localize and characterize cholecystokinin (CCK) receptor binding sites in trigeminal and dorsal root ganglia, and in the spinal cord of the rat, rabbit, and monkey. In the rabbit and monkey, a substantial number, 90 +/- 21% and 24 +/- 8%, respectively, of trigeminal and dorsal root ganglion neurons express CCK binding sites. In the spinal cord, the highest concentration of CCK receptors is found in laminae I and II, which is the major termination site of dorsal root ganglia neurons expressing CCK receptor binding sites. Neonatal capsaicin treatment of the rat results in a 70% decline in CCK receptor binding sites in laminae I and II of the spinal cord, indicating that dorsal root ganglia neurons are a major source of CCK receptors in the spinal cord. Pharmacological experiments using selective CCK-A and CCK-B receptor antagonists demonstrate that CCK-B is the prominent CCK receptor subtype in trigeminal and dorsal root ganglia neurons in the rat, rabbit, and monkey. In the rat and rabbit spinal cord, CCK-B binding sites are the prominent subtype, whereas in the monkey cord, CCK-A is the prominent receptor subtype. These results demonstrate that CCK-B receptors are expressed by a substantial percentage of dorsal root ganglion neurons at all spinal levels, and that CCK may antagonize opiate analgesia at the level of the primary afferent neuron itself.     

Detection of a cell surface antigen found on rat peripheral nervous system neurons and multiple glia: Astrocytes,oligodendrocytes, and Schwann cells

A cell surface component has been identified that is found on cultured rat dorsal root ganglion neurons and Schwann cells and also cultured brain astrocytes and oligodendrocytes. This component was detected with a monoclonal antibody originally generated to the NG108 (N18 mouse neuroblastoma × C6 rat astrocytoma) hybrid cell line. The antibody, designated B2C11, binds to cultured peripheral nervous system cells: intact dorsal root ganglion and trigeminal neurons and cultured dorsal root ganglion and sciatic nerve Schwann cells. The binding of B2C11 to dorsal root ganglion neurons in vivo was confirmed by immunofluorescence analysis of cryostat sections. However, cultured embryonic rat central neurons showed no detectable binding of B2C11. Cultured brain cells containing glial fibrillary acidic protein (astrocytes) and also oligodendrocytes cultured from corpus collosum did bind B2C11 on their surfaces. B2C11 immunoprecipitation of detergent-solubilized membrane proteins from both lactoperoxidase iodinated C6 and PC12 rat pheochromocytoma cells indicated a single band with an apparent molecular weight of 21,000–23,000. Analysis of B2C11 binding to particulate protein preparations from adult rat organs showed highest specific activity in dorsal root ganglia. Other neural tissues had substantial binding. Some nonneural tissues (lung, kidney, and small intestine) expressed significant antigen levels, whereas others (heart, liver, and skeletal muscle) had a B2C11 antigen-specific activity less than 5% of that of dorsal root ganglia. Thus the B2C11 antigen is enriched in neural tissues, where it is found on the surfaces of a unique set of neuronal and glial cells.     

A quantitative study of retrograde axonal transport in motor and sensory neurons

We used 3H N-succinimidyl propionate to covalently label in vivo proteins of the rat sciatic nerve, and studied the accumulation of radioactively labeled proteins in the cell bodies of the ipsilateral dorsal root ganglion and ventral horn of spinal cord to assess retrograde axonal transport in sensory and motor neurons respectively. In each case the early accumulation of a small amount of radioactively labeled protein is followed by the later accumulation of a larger amount, which subsequently declines to lower levels. The differences between accumulation in the motor neuron and sensory neuron are discussed. Quantitative assessment of retrograde axonal transport will allow future determination of alterations in that transport after nerve injury and in toxic states, which will help elucidate the role of retrogradely transported proteins in neuronal cell biology.     

Nerve growth factor modulates the activation status and fast axonal transport of ERK 1/2 in adult nociceptive neurones

Mature dorsal root ganglion cells respond to neurotrophins, and the intracellular signalling pathways activated by neurotrophins have been characterized in vitro. We have now used immunocytochemistry and Western blots to examine the expression and activation of extracellular signal-regulated protein kinase-1/2 (ERK) in rat dorsal root ganglion cells in vivo, using antisera to total (tERK) and phosphorylated (pERK) forms. This has revealed a number of novel findings. tERK immunoreactivity is present in most dorsal root ganglion cells but is expressed most strongly in small (nociceptive) cells and, surprisingly, is absent in a population of large cells that expressed trkB or trkC but mainly lack p75(NTR) immunoreactivity. In contrast pERK is prominent in a few trkA cells and in satellite glial cells, and is further increased by NGF treatment. tERK and pERK both undergo fast anterograde and retrograde axonal transport, indicated by accumulation at a sciatic nerve ligature, and NGF reduces the level of retrograde pERK transport.     

Type-1 angiotensin receptors are expressed and transported in motor and sensory axons of rat sciatic nerves

Angiotensin II (Ang II) and its type-1 receptor (AT₁) occur in neurons at multiple locations within the organism, but the basic biology of the receptor in the nervous system remains incompletely understood. We previously observed abundant AT₁-like binding sites and intense expression of AT₁ immunoreactivity in perikarya of the dorsal root ganglion and ventral horn of the rat spinal cord. We have now examined the receptor in rat sciatic nerve, including the dynamics of its axonal transport. Ligand-binding autoradiography of resting nerve showed “hot spots” of ¹²⁵I-Ang II binding that could be specifically blocked by the AT₁ antagonist, losartan. Immunohistochemistry with an AT₁-antibody validated by Western blots also showed patches of AT₁-reactivity in nerve. These patches were localized around large myelinated axons with faint immunoreactivity in their lumens. Sixteen hours after nerve ligation there was no change in the patches or hot spots, but luminal AT₁-reactivity increased dramatically in a narrow zone immediately above the ligature. With double ligation there was a pronounced accumulation of AT₁ immunoreactivity proximal to the upstream ligature and a very slight accumulation distal to the second ligature. This asymmetric pattern of accumulation, confirmed by quantitative receptor binding autoradiography, probably reflected axonal transport rather than local production of receptor. Retrograde tracing and stereological analysis to determine the source of transported AT₁ indicated that many AT₁-positive fibers arise in the ventral horn, and a larger number arise in dorsal root ganglia. A corresponding result was obtained with double-label immunohistochemistry of ligated nerve, which showed AT₁ accumulations in both motor and sensory fibers. We conclude that somatic sensory and motor neurons of the rat export substantial quantities of AT₁ into axons, which transport them to the periphery. The physiologic implications of this finding require further investigation.     

Neuropeptide Y modulates neurotransmitter release and Ca2+ currents in rat sensory neurons

Using 125I-labeled neuropeptide Y (NPY) and peptide YY (PYY), we demonstrated the existence of specific receptors for these peptides on rat dorsal root ganglion (DRG) cells grown in primary culture. Scatchard analysis of membrane homogenates indicated that the peptides bound to 2 populations of sites, with approximate affinities of 0.08 and 6.5 nM. Only low levels of binding were detected on sympathetic neurons cultured from the same animals or on a variety of neuronal clonal cell lines. The binding of 125I-NPY and 125I-PYY to DRG cell membranes was considerably reduced by the nonhydrolyzable analog of GTP, Gpp(NH)p. The major effect of Gpp(NH)p was to reduce the number of lower-affinity NPY binding sites without altering the number of high-affinity binding sites. NPY potently inhibited Ca2+ currents recorded under voltage clamp in rat DRG cells. Both the transient and sustained portions of the Ca2+ current were inhibited. The inhibitory effects of NPY were completely blocked following treatment of the cells with pertussis toxin. Depolarization elicited a large influx of Ca2+ into DRG neurons as assessed using fura-2-based microspectrofluorimetry. This influx of Ca2+ could be partially inhibited by NPY. Furthermore, NPY effectively inhibited the depolarization-induced release of substance P from DRG cells in vitro. Thus, NPY may be an important regulator of sensory neuron function in vivo.     

Suppression of neurite outgrowth by high-dose nerve growth factor is independent of functional p75NTR receptors

We have previously demonstrated that high concentrations of nerve growth factor suppress neurite outgrowth from sensory neurons. Inhibition could be mediated by either the p75NTR or TrkA receptor. We used a functional block of p75NTR by REX antibody in rat dorsal root ganglion neurons and dorsal root ganglion cultures from p75NTR knockout mice. In both systems, high-dose NGF inhibited neurite outgrowth, implying that p75NTR is not involved in suppression of neurite outgrowth. Confocal images of dissociated dorsal root ganglion neurons exposed to fluorescence-tagged NGF showed ligand internalization. Radioligand binding indicated disappearance of high-affinity binding sites from the surface of dorsal root ganglia after treatment with 200 ng/ml NGF for 1 h. Downstream signaling showed sustained hyperphosphorylation of MAPK (Erk(1-2)) but not of SNT or Akt. High-dose NGF may induce cytoplasmic relocation of the receptor TrkA and axonal growth arrest independently of p75NTR.     

Branching of sensory axons in the dorsal root and evidence for the absence of dorsal root efferent fibers

The present study demonstrates that there are more dorsal root axons than dorsal root ganglion cells in the L₆-S₁ dorsal roots of the rat. The excess fibers do not come from aberrant dorsal root ganglion cells and our control procedures indicate that there are no extraneous fibers in these dorsal roots. Accordingly many dorsal root axons must branch in the dorsal root.     

周围神经损伤后脊神经节感觉神经元胞体形态学的变化

目的 研究周围神经损伤后脊神经节感觉神经元胞体形态学的变化以探讨其主要死广性质。方法 切断并原位吻合大鼠右侧坐骨神经，左侧不作任何处理，作为对照；于术后不同时间取L4-L6脊神经节作光镜和电镜观察，观察脊神经节感觉神经元胞体形态的变化。结果 光镜下，损伤的脊神经节感觉神经元胞体染色质浓染；电镜下，细胞膜内陷，分割细胞内容物成凋亡小体；而对侧脊神经节感觉神经元胞体均一、无变化。结论 大鼠坐骨神经损伤后，脊神经节感觉神经元有死亡，其胞体的形态学变化符合细胞凋亡特征。     

Effects of gonadal steroids on nerve growth factor receptors in sympathetic and sensory ganglia of neonatal rats

The numbers of neurons in the rat superior cervical sympathetic ganglion (SCG) differ in males and females, with the males having 30% more SCG neurons than females at 60 days of age. This sex difference arises during the early postnatal period, when testosterone administration increases the numbers of neurons and alters the nerve growth factor (NGF) content of the rat SCG. In contrast, there is no gender difference in number of neurons in the L1 dorsal root ganglion. In both males and females, the amount of NGF bound per ganglion increased between postnatal days 5 and 15 (P5 and P15) in both dorsal root ganglia (DRGs) and the SCG. There is also a gender difference in NGF binding: SCGs and DRGs of female rats at both P5 and P15 bind more NGF per ganglion than do those of males. This effect was more marked in DRGs than in the SCG. Treatment of neonatal females with testosterone reduced NGF binding in both SCGs and DRGs to levels comparable to males at P5, and in DRGs at P15. In contrast, treatment of males with testosterone from birth resulted in a 2-3 fold increase of NGF binding in both SCGs and DRGs as compared to controls at P15. At P15, testosterone treatment of females increased NGF binding in the SCG. Males and females had opposing responses to neonatal exposure to estradiol. Treatment with estradiol from birth increased NGF binding in SCGs and DRGs of females, but had no effect on NGF binding of SCGs, and reduced NGF binding in DRGs of males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preferential loss of large lumbar primary sensory neurons in carcinomatous sensory neuropathy

Morphometric studies at autopsy of a patient with sensory neuropathy associated with small-cell lung carcinoma showed preferential loss of large-diameter sensory nerve cell bodies (fifth lumbar dorsal root ganglion), marked decrease of large myelinated fibers in the dorsal root and sural nerve, and almost total loss of myelinated fibers in the fasciculus gracilis.     

In vivo ANTI-NGF induces sprouting of sensory axons in dorsal roots

Newborn rats were given subcutaneous injections of antibodies to mouse beta -NGF (ANTI-NGF) daily for 1 month. The number of neurons in T4-T6 dorsal root ganglia (DRG) and the numbers of myelinated and unmyelinated axons in the dorsal roots of the same segments were counted in the ANTI-NGF animals and in normal littermates. The ANTI-NGF rats had 38% fewer neurons in thoracic ganglia but 17% more myelinated and 40% more unmyelinated fibers than their untreated littermates. Dorsal root ganglion cells also have a larger average size in the ANTI-NGF animals, which we interpret as a disproportionate loss of small cells. These data are interpreted as showing that some dorsal root ganglion cells, principally small ones, die when endogenous NGF is inactivated, and that the remaining cells emit more processes than normal. Thus, removal of NGF has what appears to be a paradoxical effect, a reduction in dorsal root ganglion cell numbers but an increase in dorsal root axon numbers. The relation of myelin thickness to fiber diameter is also altered, with small fibers being more thinly myelinated in the ANTI-NGF group. Thus, Schwann cell-neuronal interactions are also affected by inactivation of NGF.     

Branching of sensory axons in the peripheral nerve of the rat

The currently accepted concept of a primary sensory cell is a cell that gives rise to a central process which passes through the dorsal root to the spinal cord and a peripheral process which passes to the periphery via a peripheral nerve. If this is correct, then there should be equal numbers of sensory axons in the dorsal root, dorsal root ganglion cells, and sensory axons in the proximal peripheral nerve. The present study obtains these counts in animals in which extraneous axons have been removed from the peripheral nerve and root. The counts indicate that there are approximately 2.3 sensory axons in the dorsal root and proximal peripheral nerve for each ganglion cell in the sacral segments of the rat. We interpret these data as indicating that there is significant branching of sensory axons in the dorsal root and proximal peripheral nerve and thus the generally accepted picture of a dorsal root ganglion cell is not correct for some, perhaps all, of these cells. We offer the speculation that this peripheral branching may be an indication of single sensory neurons having receptive fields in two separate locations, and thus this may be an anatomical explanation for certain types of referred pain.     

THE EFFECT OF NALOXONE AND MORPHINE ON 3H-1-LYSINE IN VIVO INCORPORATION INTO VARIOUS NEURONS SEPARATED FROM FIXED RAT BRAIN PREPARATIONS BY ULTRASONIFICATION: A COMPARISON OF MORPHINE EFFECTS BETWEEN WISTAR AND SPRAGUE-DAWLEY RATS

A procedure is described where by ultrasonification one can separate large neurons from their surrounding neuropil from either unfixed brain and peripheral ganglion or from similar tissue fixed in 10 per cent neutral formalin for prolonged periods. The availability of such a technique permits one to readily assess the accumulation of ³H-labeled protein precursors into a wide variety of neurons, utilizing standard liquid scintillation techniques. The separation technique has been applied in this report to determine the effects of morphine, morphine plus naloxone, naloxone given alone and saline on the accumulation of ³H-1-lysine into ventral horn, Purkinje and dorsal root ganglion neurons in Sprague-Dawley rats. The data from the control and morphine-treated animals has then been compared with similar data previously obtained from Wistar rats. In Sprague-Dawley rats, morphine had no effect on ³H-1-lysine accumulation into ventral horn neurons and stimulated accumulation into Purkinje and dorsal root ganglion neurons. Naloxone stimulated lysine accumulation into dorsal root ganglion and ventral horn neurons, but had equivocal effects on Purkinje neuron ³H-lysine accumulation. When Wistar and Sprague-Dawley rats were compared, marked differences in the effect which morphine had on lysine accumulation into neurons were noted between the two strains of rat. Ventral horn and dorsal root ganglion neurons from Wistar rats had markedly higher levels of accumulation in both control and morphine-treated rats than were observed in the Sprague-Dawley animals. With Purkinje neurons, accumulation levels between the two strains overlapped each other. Morphine inhibited lysine accumulation in Wistar Purkinje neurons but stimulated it in the Sprague-Dawley animals. The profiles of the accumulation curves from two rat strains suggest that there are not only differences in rates of uptake of ³H-lysine into protein followed by degradation between various types of neurons, but differences between the two strains as well.     

Reciprocal actions of interleukin-6 and brain-derived neurotrophic factor on rat and mouse primary sensory neurons

In low-density, serum-free cultures of neurons from embryonic rat dorsal root ganglia, interleukin-6 supports the survival of less than one third of the neurons yet virtually all of them bear interleukin-6 alpha-receptors. A finding that might explain this selectivity is that interleukin-6 acts on sensory neurons in culture through a mechanism requiring endogenous brain-derived neurotrophic factor. Antibodies or a trkB fusion protein that block the biological activity of brain-derived neurotrophic factor synthesized by dorsal root ganglion neurons also block the survival-promoting actions of interleukin-6 on these neurons. Two results indicate that interleukin-6 influences synthesis of brain-derived neurotrophic factor in adult dorsal root ganglion neurons. Intrathecal infusion of interleukin-6 in rats increases the concentration of brain-derived neurotrophic factor mRNA in rat lumbar dorsal root ganglia. The induction of brain-derived neurotrophic factor in dorsal root ganglion neurons that is seen after nerve injury in rats or wild-type mice is severely attenuated in mice with null mutation of the interleukin-6 gene. In brief, the ability of interleukin-6 to support the survival of embryonic sensory neurons in vitro depends upon the presence of endogenous brain-derived neurotrophic factor and the induction of brain-derived neurotrophic factor in injured adult sensory neurons depends upon the presence of endogenous interleukin-6.     

Relationships between capsaicin sensitivity of mammalian sensory neurons, cell size and type of voltage gated Ca-currents.

(1) The relationships between capsaicin sensitivity, cell size and the expression of voltage-dependent high-threshold (L- and N-type) and low-threshold (T-type) Ca-currents were investigated in cultured adult rat dorsal root ganglion neurons using the whole-cell patch-clamp technique. (2) The capsaicin (10 microM) evoked inward current was used to identify the capsaicin-sensitive population. To identify the voltage-dependent Ca-currents, the activation threshold and the inactivation kinetic were used. (3) In cultured dorsal root ganglion cells the capsaicin sensitivity is not restricted to the small cell population. About 28% of cells with large cross sectional areas (greater than or equal to 1001 microns 2) responded with about the same amplitude per micron2 cross sectional area as small cells. (4) The amplitude of the capsaicin evoked inward current depended on the length of time the cells were maintained in culture. (5) All cells exhibited the high-threshold Ca-currents. About 45% of the capsaicin-insensitive cells also had low-threshold Ca-currents. None of the capsaicin-sensitive cells had the low-threshold type. (6) Most of the cells which expressed the low-threshold type were in the upper size range.     

Correlation between GAP43 and nerve growth factor receptors in rat sensory neurons

In mature rat sensory neurons, expression of the gene for the growth-associated protein, GAP43, was studied by in situ hybridization with a cDNA probe. Among neurons in normal lumbar dorsal root ganglia, labeling for GAP43 mRNA was heterogeneous, approximately one-half of the neurons being densely labeled. To characterize the latter population, individual neurons were examined in adjacent sections processed either for GAP43 hybridization or NGF-receptor radioautography. Virtually all neurons with high-affinity NGF binding sites had high basal levels of GAP43 mRNA and most GAP43-positive neurons bore NGF receptors. Another NGF-responsive population, sympathetic neurons in the superior cervical ganglion, also had high basal concentrations of GAP43 mRNA. Further co-localization studies in dorsal root ganglia were performed with immunohistochemistry for somatostatin and enzyme histochemistry for acid phosphatase. The latter 2 groups of sensory neurons have been previously shown to lack high-affinity receptors and were here shown to have low basal concentrations of GAP43 mRNA. From this and earlier studies, it can be assumed that substance P-immunoreactive neurons and strongly positive CGRP neurons synthesize GAP43 at high basal rate. One week following peripheral nerve transection, almost all neurons had high concentrations of GAP43 mRNA without correlation with NGF binding. Intrathecal infusion of NGF after the sciatic nerve was cut did not strongly influence this post-traumatic elevation in GAP mRNA. In normal dorsal root ganglia, neurons that have high-affinity NGF binding sites and are therefore potentially responsive to NGF also have high basal rates of synthesis of GAP43.(ABSTRACT TRUNCATED AT 250 WORDS)