Effect of Peripheral Axotomy on Expression of Neuropeptide Y Receptor mRNA in Rat Lumbar Dorsal Root Ganglia

Using in situ hybridization, the expression of the mRNA for a neuropeptide Y (NPY) receptor, was studied in lumbar (L) 4 and 5 dorsal root ganglia (DRGs) of normal rats and at various intervals after unilateral sciatic nerve transection. Twenty percent of all normal DRG neurons were NPY receptor mRNA-positive, and the majority of these neurons were of the small type, with only a few labelled medium-sized and large neurons. In L5 normal ganglia NPY receptor mRNA colocalized with substance P, calcitonin gene-related peptide and galanin mRNAs in small neurons, but not in medium-sized or large neurons containing these peptides. NPY receptor mRNA was not observed in somatostatin or nitric oxide synthase mRNA-positive neurons. Sciatic nerve transection induced a marked decrease in NPY receptor mRNA levels. However, in parallel there was a transient increase in the number of NPY receptor mRNA-positive small neuron profiles, but the intensity of labelling was mostly very low, although a few strongly labelled, small neuron profiles were also encountered. In addition, axotomy caused a marked increase in the number of NPY receptor mRNA-positive large neuron profiles in the ipsilateral DRGs, and they constituted 15–20% of counted DRG neuron profiles and 45–65% of counted large neuron profiles, 7–28 days after axotomy. In L5 DRGs, ipsilateral to the axotomy, NPY receptor mRNA colocalized with NPY mRNA in many large and some medium-sized neuron profiles, with galanin mRNA in some small, medium-sized and large neuron profiles and with vasoactive intestinal polypeptide mRNA in some small and medium-sized neuron profiles and a few large profiles. Occasionally, NPY receptor mRNA was observed in nitric oxide synthase mRNA-positive small neurons. In the dorsal horn, NPY receptor mRNA-positive small neurons were concentrated in lamina II at L4 and L5 levels, and were scattered in deeper laminae. No marked changes were observed ipsilateral to the axotomy. No NPY receptor mRNA-positive cells were found in the normal rat gracile nucleus, or in this nucleus after axotomy. These results show that a NPY receptor may be a prejunctional receptor in primary afferent neurons and play a role in the modulation of somatosensatory information, both in normal and lesioned primary afferent DRG cells. However, axotomy induced a distinct shift in NPY receptor mRNA expression from small to large neurons, indicating that sensitivity to NPY is switched from one modality to another. Thus, not only several sensory neuropeptides, as shown in previous studies, but at least also one of the peptide receptors change their expression dramatically in response to axotomy, suggesting complex adaptive responses.     

Neuropeptides in dissociated cell cultures of mammalian spinal cord and dorsal root ganglion

Immunohistochemical studies of leucine-enkephalin, somatostatin, vasoactive intestinal polypeptide and neurotensin were carried out in dissociated cell co-cultures of embryonic mouse spinal cord and dorsal root ganglion, using the peroxidase-antiperoxidase technique. Leucine-enkephalin immunoreactivity exceeded that of the other peptides in these coculture preparations. Leucine-enkephalin, substance P and somatostatin were also studied in spinal cord cultures (without dorsal root ganglia) and in dorsal root ganglia cultures (without spinal cord). Each of these peptides was present in only a small percentage (<10%) of perikarya and processes in spinal cord cultures. No leucine-enkephalin immunoreactivity was seen in dorsal root ganglion cultures; a considerable proportion of the processes were immunoreactive for substance P or somatostatin. These observations suggest that co-cultures of spinal cord and dorsal root ganglia can provide a simplified in vitro “model” of the nervous system for the study of peptidergic interactions.     

Cholecystokinin in Mammalian Primary Sensory Neurons and Spinal Cord: In Situ Hybridization Studies in Rat and Monkey

The peptide cholecystokinin (CCK) has been suggested to be involved in nociception, but its exact localization at the level of the spinal cord and in spinal ganglia has been a controversial issue. Therefore the distribution of messenger RNA (mRNA) for CCK was studied by in situ hybridization using oligonucleotide probes on sections of adult rat lumbar dorsal root ganglia following unilateral section of the sciatic nerve and on sections of untreated monkey trigeminal ganglia, spinal cord and spinal ganglia from all levels. For comparison, calcitonin gene-related peptide (CGRP) mRNA was also studied in the monkey tissue using the same techniques. Peripheral sectioning of the sciatic nerve in the rat resulted in the appearance of detectable CCK mRNA in up to 30% of remaining ipsilateral L4 and L5 dorsal root ganglion neurons 3 weeks after surgery, with a distinct but more limited appearance also in the contralateral ganglia. No cells, or only single cells, could be seen in normal control rat ganglia. In contrast, in the normal monkey, ∼20% of dorsal root ganglion neurons, regardless of spinal level, and 10% of trigeminal ganglia neurons expressed mRNA for CCK. CGRP mRNA was expressed at detectable levels in ∼80% of these monkey dorsal root ganglion neurons. In the monkey spinal cord, CCK mRNA was detected in the dorsal horn and in motoneurons, whereas CGRP mRNA was only seen in motoneurons. The present results suggest that CCK peptides can be involved in sensory processing in the dorsal horn of the spinal cord in normal monkeys and in rats after peripheral nerve injury, adding one more possible excitatory peptide to the group of mediators in the dorsal horn.     

Galanin Receptor Binding Sites in Adult Rat Spinal Cord Respond Differentially to Neonatal Capsaicin, Dorsal Rhizotomy and Peripheral Axotomy

The discrete distribution and possible changes in specific [¹²⁵I]galanin binding sites were evaluated in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. The highest density of [¹²⁵I]galanin binding sites in the normal rat spinal cord was particularly evident in the superficial layers of the dorsal horn whereas moderate to low amounts of labelling were associated with the deeper dorsal horn, areas around the central canal and the ventral horn. Capsaicin-treated rats, compared to littermate controls, showed a significant bilateral increase in [¹²⁵I]galanin binding in the superficial laminae of the dorsal horn. Similarly, unilateral dorsal rhizotomy evoked a significant increase in the density of [¹²⁵I]galanin binding sites in the superficial dorsal horn ipsilateral to surgery. Section of the sciatic nerve, on the other hand, induced a significant depletion in [¹²⁵I]galanin binding in laminae I and II of the ipsilateral dorsal horn. These results, in parallel to those reported for galanin immunoreactivity under similar conditions, suggest that [¹²⁵I]galanin binding sites are preferentially located postsynaptically to the primary afferent fibre terminals in the dorsal horn of the spinal cord. Thus it seems that galanin, at the level of the dorsal spinal cord, regulates the processing of nociceptive information by acting on its own class of specific receptors located postsynaptically to primary sensory terminals.     

Substance P-, somatostatin-, vasoactive intestinal peptide- and cholecystokinin-like levels in the spinal cord of polyarthritic rats

Substance P-, somatostatin-, vasoactive intestinal polypeptide- and cholecystokinin-like levels were measured in lumbar dorsal and ventral cord of polyarthritic rats and compared with those obtained in vehicle-treated rats taken as controls. Polyarthritis decreased substance P concentration in lumbar ventral cord and increased cholecystokinin level in lumbar dorsal cord, while the other two peptides did not show any change. The results are discussed in relation to immunohistochemical data found in the literature.     

Innervation of Putative Rapidly Adapting Mechanoreceptors by Calbindin- and Calretinin-immunoreactive Primary Sensory Neurons in the Rat

Calbindin and calretinin are two homologous calcium-binding proteins that are expressed by subpopulations of primary sensory neurons. In the present work, we have studied the distribution of the neurons expressing calbindin and calretinin in dorsal root ganglia of the rat and their peripheral projections. Calbindin and calretinin immunoreactivities were expressed by subpopulations of large- and small-sized primary sensory neurons and colocalized in a majority of large-sized ones. The axons emerging from calbindin- or calretinin-immunoreactive neurons innervated muscle spindles, Pacini corpuscles and subepidermal lamellar corpuscles in the glabrous skin, formed palisades of lanceolate endings around hairs and vibrissae, and gave rise to intraepidermal nerve endings in the digital skin. Since most of these afferents are considered as rapidly adapting mechanoreceptors, it is concluded that calbindin- or calretinin-expressing neurons innervate particular mechanoreceptors that display physiological characteristics of rapid adaptation to stimuli.     

Activation by Cutaneous or Visceral Noxious Stimulation of Spinal Neurons Projecting to the Medullary Dorsal Reticular Nucleus in the Rat: A c-fos Study

The involvement of spinal neurons in the transmission of cutaneous and visceral nociceptive input to the medullary dorsal reticular nucleus was studied. Rats were injected with cholera toxin subunit B in the left dorsal reticular nucleus and subjected 4 days later to noxious mechanical, thermal or chemical stimulation of the proximal internal aspect of the left thigh, or to chemical stimulation of the urinary bladder. Sections of spinal segments T₁₃-L₃ were processed immunocytochemically for cholera toxin subunit B and Fos protein. The percentage of double-labelled cells in the population of Fos-positive cells was higher in lamina I (1–4%) than in deeper laminae (0–0.7%) following all stimuli. The percentage of double-labelled cells in the population of retrogradely labelled cells was 30–53% in lamina I and 0–5% in laminae III—X. Visceral stimulation activated more retrogradely labelled lamina I cells than any kind of cutaneous stimulation. Pyramidal cells were activated in higher numbers than multipolar and flattened cells after thermal cutaneous or visceral stimulation, and in lower numbers than multipolar cells after mechanical stimulation. These results suggest that, in the experimental conditions used, spinal cord cells conveying noxious input to the dorsal reticular nucleus are concentrated in lamina I. They further indicate that the spinaldorsal reticular nucleus pathway plays a major role in the transmission of nociceptive visceral input, and point to the preferential involvement of pyramidal cells in cutaneous thermal and visceral processing.     

Implication of a Nitric Oxide Synthase Mechanism in the Action of Substance P: L-NAME Blocks Thermal Hyperalgesia Induced by Endogenous and Exogenous Substance P in the Rat

The effects of i.p. administration of the nitric oxide synthase inhibitor N^G-nitro-L-arginine methylester (L-NAME) and its inactive isomer, D-NAME, were tested in two nociceptive paradigms in the rat. In the first paradigm, rats were lightly anaesthetized with a mixture of chloral hydrate (120 mg/kg, i.p.) and sodium pentobarbital (20 mg/ kg, i.p.). Tail flick reaction times were monitored and thermal hyperalgesia was induced by immersion of the tail in hot water at 55°C for 1.5 min. In the groups of rats pretreated with saline (n= 5), 100 mg/kg D-NAME (n= 6), 10 (n= 5) or 25 (n= 6) mg/kg L-NAME, this thermal injury induced a transient reduction in the reaction time that was 54–59% of the baseline value. However, in the groups of rats pretreated with 50 (n= 6) or 100 (n= 7) mg/kg L-NAME the reaction times were 73.9 ± 2.7% (P < 0.05) and 102.3 ± 0.9% (P < 0.001) of the baseline values respectively, indicating a block of the hyperalgesic responses seen in the other groups. As this hyperalgesia has been reported to be blocked by NK-1 receptor antagonists, it is suggested that it is due to the action of endogenous substance P. In the second paradigm, tail flick responses were monitored in the awake rat and thermal hyperalgesia was induced by intrathecal administration of substance P (6.5 nmol) via a chronically implanted catheter. In the group of rats pretreated with saline (n= 5) or D-NAME (n= 5; 100 mg/kg), substance P reduced the reaction time to 39.1 ± 9.9 and 45.5 ± 2.1% of the baseline value respectively. However, in the rats pretreated with L-NAME (n= 6; 100 mg/kg), the reaction time following substance P administration was 108.8 ± 8.8% of the baseline value (P < 0.001), indicating a block of the hyperalgesic response induced by substance P. These data indicate that thermal hyperalgesia induced by endogenously released or exogenously administered substance P, are blocked by L-NAME but not by its enantiomer, D-NAME. Therefore an involvement of a nitric oxide synthase mechanism in the hyperalgesic responses to substance P is suggested.     

Studies on the location and release of cholecystokinin and vasoactive intestinal peptide in rat and cat spinal cord

By radioimmunoassay vasoactive intestinal peptide (VIP) and cholecystokinin (CCK) are found in the cat lumbar spinal ganglion and spinal cord with levels in dorsal greater than ventral horn. Unilateral rhizotomy, but not cervical hemisection produced a significant but incomplete depletion of CCK and VIP immunoreactivity in dorsal, but not ventral horn. Intrathecal capsaicin (0.5 mg) had no effect on the levels of spinal VIP or CCK. Intrathecal colchicine (0.5 mg)produced a significant increase in the levels of VIP in the dorsal and ventral horn but had no effect on the levels of CCK. The present experiments, using a preparation which permits in situ superfusion of the spinal cord, demonstrated in the chloralose-urethanized cat and rat the presence of measurable levels of VIP and CCK. In rats, the addition of potassium (40 mM in excess) resulted in a 138% and 46% increase in the levels of CCK and VIP, respectively above resting levels (3.7 +/- 1.2 fmol/ml/10 min and 1.7 +/- 0.5 fmol/ml/10 min, respectively). The deletion of calcium and substitution of cobalt (2 mM) resulted in a significant reduction in the potassium-evoked release. Intrathecal picrotoxin doubled the levels of CCK, but had no effect on the levels of VIP in the spinal superfusates. Capsaicin (3 X 10(-4) M) had no effect on the levels of either peptide in rat spinal superfusate. In cats, bilateral electrical stimulation of the sciatic nerve at high, but not low intensity, resulted in a 218% and 132% increase above prestimulation baseline in the levels of CCK and VIP, respectively. Separation of immunoreactivity on a Sephadex G-50 superfine column of the spinal superfusates and the extracted material from cat spinal cord, revealed that the immunoreactive CCK species in tissue co-migrated with the 8 and 33 amino acid peptide fragments. In the release samples, however, all the radioimmunoassayable activity migrated with the peak corresponding with CCK. No other peaks were detected. Column separation of spinal cord and the superfusate obtained during basal and evoked release, revealed that all activity in both the tissue and perfusate samples, travelled in a single peak which co-migrated with authentic VIP.     

Expression of c-Jun as a Response to Dorsal Root and Peripheral Nerve Section in Damaged and Adjacent Intact Primary Sensory Neurons in the Rat

It was previously shown that the immediate early gene, c-jun , was highly expressed over long periods, in both peripheral sensory and motor neurons following axon damage or block of axoplasmic transport. Here we have examined the question of whether the expression of c-Jun protein is related to axon injury per se or to the process of axon growth. We have examined dorsal root ganglion (DRG) cells subjected to different manipulations which are associated with varying degrees of regrowth, as follows: (i) after peripheral nerve section, where it appears that all damaged neurons make some regenerative effort. 1 – 24 days after sciatic nerve section and ligation most cells in L4/L5 DRG were c-Jun-positive; (ii) after section of the central processes of the DRG cells, which then showed a slow and limited regrowth of their axons towards, but not into, the spinal cord. This resulted in a variable, but significant, expression of c-Jun in a small number of DRG cells; (iii) in intact sensory neurons that were offered the opportunity to sprout into adjacent denervated peripheral tissue. The sciatic nerve was ligated and the response of cells in the L3 ganglia (many of which project to the saphenous nerve) was measured. A small but significant number of cells were c-Jun-positive; (iv) in intact sensory neurons that were offered the opportunity to sprout centrally into partialy denervated neuropil of the spinal cord. We examined neurons in the L3 DRG after rhizotomy of the adjacent L4/L5 dorsal roots. Previous work suggests that sensory neurons show at best a very limited growth under these conditions. No significant increase was seen in c-Jun expression in these cases. These results suggest that c-Jun expression is closely correlated with growth and regeneration, and not simply a consequence of neuronal injury.     

Distribution of 125I-galanin binding sites, immunoreactive galanin, and its coexistence with 5-hydroxytryptamine in the cat spinal cord: biochemical, histochemical, and experimental studies at the light and electron microscopic level.

The distribution of galanin-like immunoreactivity (GAL-LI) in the spinal cord of the cat was studied by use of indirect histochemistry and the peroxidase-antiperoxidase (PAP) technique. In the ventral horn GAL-immunoreactive (IR) axonal fibers and terminals were most frequent in the ventral part of the motor nucleus. The GAL-IR axons also contained 5-hydroxytryptamine (5-HT)-LI, and they disappeared after spinal cord transection. It was concluded that these GAL-IR fibers belong to the serotoninergic bublospinal pathway. In the medulla oblongata from normal cats, scattered GAL-IR cell bodies were encountered within the nucleus raphe obscurus and nucleus raphe pallidus. Electron microscopic observations revealed that the fine structure of the GAL-IR axonal boutons in the motor nucleus was similar to that of 5-HT-IR boutons with a varying number of immunoreactive large dense core vesicles. The postsynaptic element in all cases studied was a dendrite. A dense GAL-IR axonal plexus was found in the superficial laminae I-II of the dorsal horn. Coexistence was found between the GAL- and substance P-LI in fibers within the dorsal horn plexus. Spinal cord transection did not alter the pattern of GAL-LI in the dorsal horn, while the vast majority of GAL-IR axonal swellings disappeared following dorsal root sectioning. Electron microscopic observations in lamina II (substantia gelatinosa) revealed that the GAL-IR axonal terminals could be divided into two main groups. One with small to medium-sized axonal boutons formed synaptic contacts with both dendritic and axonal profiles. The other formed the central axon terminals of glomeruli, suggesting that GAL-LI may be present in C-type primary afferents. Numerous small GAL-IR cell bodies were encountered in laminae II and III. GAL-IR cell bodies were also observed in lamina X. The dorsal root ganglia contained a low but consistent number of small to medium-sized GAL-IR cell bodies, which all contained immunoreactive calcitonin gene-related peptide (CGRP). Following peripheral sciatic nerve transection, the number and the labeling intensity of GAL-IR cell bodies in the corresponding dorsal root ganglia showed a moderate increase. Radioimmunoassay revealed that the concentration of GAL-LI increased along the rostrocaudal axis of the normal spinal cord, and was about three times higher in the dorsal than in the ventral regions. The concentration in the dorsal root ganglia was intermediate to those seen in the corresponding dorsal and ventral cord regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Type-specific localization of monoamine oxidase in the enteric nervous system: relationship to 5-hydroxytryptamine, neuropeptides, and sympathetic nerves

The localization in the guinea pig enteric nervous system (ENS) of monoamine oxidase (MAO) types A and B was investigated at the light and electron microscopic levels. Immunocytochemistry was used to visualize the enzyme protein and histochemistry was employed to study catalytic activity. Type specificity was achieved in histochemical studies by using deprenyl (0.5 microM) to inhibit MAO-B or clorgyline (0.1 microM) to inhibit MAO-A. The distribution of MAO-B immunoreactivity in the ENS corresponded to that of the sites of MAO activity found histochemically to be inhibited by deprenyl, but not clorgyline. MAO-B was observed to be the primary type of MAO found in the intrinsic elements of the ENS and was located in subsets of neurons in both submucosal and myenteric plexuses. MAO-B was not demonstrated immunocytochemically or histochemically in enteric glia, nor, at the light microscopic level, was there significant MAO-B activity or immunoreactivity in serotonin (5-HT)-immunoreactive neuronal cell bodies. In the submucosal plexus about 50% of the neurons expressed MAO-B; these neurons also contained neuropeptide y (NPY) and/or calcitonin gene related peptide (CGRP), but not substance P or vasoactive intestinal polypeptide (VIP). About 10% of myenteric neurons were intensely reactive for MAO-B; again MAO-B was co-localized with NPY and/or CGRP. In contrast to intrinsic neurons, extrinsic CGRP-immunoreactive nerve fibers contained no demonstrable MAO activity or immunoreactivity. Moreover, the sympathetic innervation, identified as varicose axons that degenerated after administration of 6-hydroxydopamine, contained abundant MAO-A, but no MAO-B activity or immunoreactivity. It is concluded that MAO-B is characteristic of a subset of intrinsic enteric neurons, while MAO-A is confined to the sympathetic innervation, which is extrinsic. At the electron microscopic level individual cells varied greatly in their degree of immuno- or cytochemically demonstrable MAO-B, which was most concentrated on the outer membranes of mitochondria. MAO-B immunoreactivity (but not cytochemical activity) was found on mitochondria in some serotoninergic perikarya identified by the simultaneous radioautographic detection of the uptake of 3H-5-HT. Mitochondria in most serotoninergic axon terminals displayed both MAO-B activity and immunoreactivity. Neurons receiving serotoninergic synapses often, but not invariably, contained MAO-B. Inhibition of neither MAO-B nor MAO-A appeared to slow the disappearance of 3H-5-HT loaded into enteric neurons significantly, even when intraneuronal storage of 5-HT was inhibited with tetrabenazine.(ABSTRACT TRUNCATED AT 400 WORDS)     

The origins of the sciatic nerve and changes in neuropeptides after axotomy: a double labelling study using retrograde transport of Tru blue and vasoactive intestinal polypeptide immunohistochemistry

One month after sciatic nerve section, only dorsal root ganglion cells which take up True blue applied to the cut end of the nerve show vasoactive intestinal polypeptide immunoreactivity. This indicates that VIP expression is only in cells with damaged axons. Motor axons with True blue-positive perikarya in the ventral horn originate from the fourth to sixth lumbar segments whereas unmyelinated and small myelinated sensory nerves terminate in the third to fifth lumbar segments of the spinal cord.     

Electroacupuncture and A‐317491 depress the transmission of pain on primary afferent mediated by the P2X3 receptor in rats with chronic neuropathic pain states

P2X is a family of ligand‐gated ion channels that act through adenosine ATP. The P2X₃ receptor plays a key role in the transmission of neuropathic pain at peripheral and spinal sites. Electroacupuncture (EA) has been used to treat neuropathic pain effectively. To determine the role of EA in neuropathic pain mediated through the P2X₃ receptor in dorsal root ganglion neurons and the spinal cord, a chronic constriction injury (CCI) model was used. Sprague‐Dawley rats were divided into four groups: sham CCI, CCI, CCI plus contralateral EA, and CCI plus ipsilateral EA. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded. Furthermore, the expression of the P2X₃ receptor was evaluated through Western blotting and immunofluorescence. The effects of EA and A‐317491 were investigated through the whole‐cell patch‐clamp method and intrathecal administration. Our results show that the MWT and TWL of EA groups were higher than those in the CCI group, whereas the expression of the P2X₃ receptor was lower than that in the CCI group. However, no significant difference was detected between the two EA groups. EA depressed the currents created by ATP and the upregulation of the P2X₃ receptor in CCI rats. Additionally, EA was more potent in reducing mechanical allodynia and thermal hyperalgesia when combined with A‐317491 through intrathecal administration. These results show that both contralateral and ipsilateral EA might inhibit the primary afferent transmission of neuropathic pain induced through the P2X₃ receptor. In addition, EA and A‐317491 might have an additive effect in inhibiting the transmission of pain mediated by the P2X₃ receptor. © 2014 Wiley Periodicals, Inc.