Quantitative autoradiographic localization of [125I]neuropeptide Y receptor binding sites in rat spinal cord and the effects of neonatal capsaicin, dorsal rhizotomy and peripheral axotomy.

Using in vitro quantitative receptor autoradiography the present study reports on the distribution and possible changes of [125I]neuropeptide Y (NPY) binding sites in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. In control spinal cord the highest density of [125I]NPY binding sites was noticed in the superficial layers of the dorsal horn whereas low-to-moderate densities of [125I]NPY binding sites were detected in the deeper dorsal horn and in the ventral horn. In comparison with control rats, neonatally treated capsaicin rats showed a significant (P less than 0.001) bilateral decrease in [125I]NPY binding sites in the superficial layers of the dorsal horn. Unilateral dorsal rhizotomy and unilateral sciatic nerve section also exhibited a significant (P less than 0.05) depletion in [125I]NPY labeling in the superficial layers of the dorsal horn ipsilateral to the surgery. These results suggest that a certain proportion of [125I]NPY receptor sites is located on the primary afferent fibers of the superficial layers of the dorsal horn. This peptide thus could play an important role in the modulation of nociceptive transmission by acting directly on primary afferent terminals.     

Neuropeptide Y and Galanin Binding Sites in Rat and Monkev Lumbar Dorsal Root Ganalia and Spinal Cord and Effect of Peripheral Axotomy

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [¹²⁵I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I–IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae 1–11. [¹²⁵I]NPY binding was most strongly attenuated by NPY_13–36, a Y2 agonist, and partially inhibited by [Leu³¹,Pro³⁴]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY_13–36 completely abolished PYY binding, which was also reduced by [Leu,³¹ Pro³⁴] NPY. However, the increase in PYY binding seen in laminae I–IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu³¹, Pro³⁴] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I–IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).     

Plasticity of &mgr; and delta Opioid Receptors in the Superficial Dorsal Horn of the Adult Rat Spinal Cord Following Dorsal Rhizotomies: A Quantitative Autoradiographic Study

The aim was to study the regulation of μ and δ opioid binding sites in the superficial layers (laminae I–II) of the dorsal horn of the adult rat spinal cord 1, 2, 4 and 12 weeks after unilateral dorsal rhizotomies of various extents. Using quantitative autoradiography and highly selective tritiated opioid ligands, we have shown that the decrease in [³H]Tyr*- d -Ala-Gly-NMe-Phe-Gly-ol ([³H]DAMGO) (μ sites) and [³H]Tyr*- d -Thr-Gly-Phe-Leu-Thr ([³H]DTLET) (δ sites) binding in the side ipsilateral to the lesion as compared to the intact side is related to the number of dorsal roots cut. In the segment central to the lesion, 1 week after the lesion, ipsilateral/contralateral side binding ratios for [³H]DAMGO were 0.70, 0.49, 0.36 and 0.25 when 1, 3, 5 and 7 roots respectively were sectioned. For [³H]DTLET, the ratios were 0.71, 0.54, 0.42 and 0.39. The time-related analysis of binding ratios showed that, in partially deafferented spinal segments after long-term deafferentation (12 weeks postlesion) there were greater numbers of μ and δ binding sites than in cases of short-term deafferentation (1–2 weeks). By contrast, in spinal segments considered as completely deafferented, there was no difference in the remaining μ and δ binding sites at 12 weeks as compared to 1 week postlesion. Consequently, it is deduced that the partial recovery of μ and δ binding observed after long-term partial deafferentation could be associated with neuronal plasticity (probably collateral sprouting) of fine diameter primary afferent fibres arising from intact dorsal roots.     

Effects of dorsal rhizotomy on neurokinin receptor sub-types in the rat spinal cord: a quantitative autoradiographic study.

Although abundant evidence suggests a major role for substance P (SP) and other neurokinins (NK) in the transmission of nociceptive information, it is not known whether the various NK receptor classes are differentially located in the substantia gelatinosa of the spinal cord where primary afferent fibres mostly terminate. In order to investigate this issue, we studied the effects of unilateral dorsal rhizotomy on binding of 125I-Bolton-Hunter-SP, (2-[125I]iodohistidyl1)-neurokinin A, and 125I-Bolton-Hunter-eledoisin as respective radioligands for the NK-1, NK-2 and NK-3 receptor sub-types. Seven, 14, 21 and 28 days following unilateral lumbosacral dorsal horn deafferentiation, NK receptor binding parameters were evaluated using quantitative receptor autoradiography. Rhizotomy produced an increase in the densities of NK-1, NK-2 and NK-3 binding sites in the superficial laminae of the dorsal horn. Increases were maximal at 14 days, post-operatively, for both NK-1 and NK-2 sites; slight recovery being observed thereafter. For NK-3 sites, unilateral rhizotomy induced a progressive increase in binding without evidence of recovery over time, at least up to 28 days post-lesion. NK-1 receptor binding parameters around the central canal and in the ventral horn were not affected by the dorsal rhizotomy. These data suggest that all 3 NK receptor classes are located post-synaptically to afferent fiber terminals in laminae I, II and X of the dorsal horn of the spinal cord.     

Quantitative autoradiographic distribution of calcitonin gene-related peptide (hCGRP alpha) binding sites in the rat and monkey spinal cord.

Calcitonin gene-related peptide (CGRP) has been implicated in various spinal functions on the basis of its presence in the substantia gelatinosa and motoneurons and the biological effects induced by intrathecal CGRP injections. We investigated here the comparative distribution of [125I]hCGRP alpha binding sites in various segments of the rat and monkey spinal cord. The immunocytochemical localization of CGRP-like material in rat spinal cord was also evaluated for comparison. In the rat spinal cord, high densities of [125I]hCGRP alpha binding sites were observed in lamina I, in a U-shaped band that included lamina X and the medial parts of laminae III-IV and in the intermediolateral and intermediomedial nuclei. The substantia gelatinosa (lamina II) contained relatively lower, but still significant, densities of [125I]hCGRP alpha binding sites, while the ventral horn showed low amounts of specific labeling. CGRP-like immunoreactive fibers, on the other hand, were heavily concentrated in laminae I-II and in the reticulated portion of lamina V of the dorsal horn. Immunoreactivity to CGRP antiserum was also noted in fibers around the central canal and in a number of motoneurons of the ventral horn. In the monkey spinal cord, [125I]hCGRP alpha binding sites were present in lamina I in a U-shaped band that included lamina X and the medial portions of laminae V-VI. Relatively low levels of [125I]hCGRP alpha binding were detected in laminae II to IV of the dorsal horn, while the ventral horn was more enriched with specific [125I]hCGRP alpha binding sites. Thus, it appears that the autoradiographic distribution of [125I]hCGRP alpha sites is species dependent in the spinal cord. Additionally, some differences are observed between the localization of [125I]hCGRP alpha binding sites and immunoreactive material in the rat spinal cord. These differences may be relevant to the purported roles of CGRP-like peptides in spinal functions such as nociception, control of sympathetic output, and motor control.     

Distribution of GLP-1 Binding Sites in the Rat Brain: Evidence that Exendin-4 is a Ligand of Brain GLP-1 Binding Sites

The distribution and biochemical properties of glucagon-like peptide (GLP)-1(7–36)amide (GLP-1) binding sites in the rat brain were investigated. By receptor autoradiography of tissue sections, the highest densities of [¹²⁵I]GLP-1 binding sites were identified in the lateral septum, the subfornical organ (SFO), the thalamus, the hypothalamus, the interpenduncular nucleus, the posterodorsal tegmental nucleus, the area postrema (AP), the inferior olive and the nucleus of the solitary tract (NTS). Binding studies with [¹²⁵I][Tyr39]exendin-4, a GLP-1 receptor agonist, showed an identical distribution pattern of binding sites. Binding specificity and affinity was investigated using sections of the brainstem containing the NTS. Binding of [¹²⁵I]GLP-1 to the NTS was inhibited concentration-dependently by unlabelled GLP-1 and [Tyr39]exendin-4 with K ₁ values of 3.5 and 9.4 nM respectively. Cross-linking of hypothalamic membranes with [¹²⁵I]GLP-1 or [¹²⁵I][Tyr39]exendin-4 identified a single ligand-binding protein complex with a molecular mass of 63 000 Da. The fact that no GLP-1 binding sites were detected in the cortex but that they were detected in the phylogenetically oldest parts of the brain emphasizes that GLP-1 may be involved in the regulation of vital functions. In conclusion, the biochemical data support the idea that the central GLP-1 receptor resembles the peripheral GLP-1 receptor. Furthermore, the presence of GLP-1 binding sites in the circumventricular organs suggests that these may be receptors which act as the target for both peripheral blood-borne GLP-1 and GLP-1 in the nervous system.     

Changes in [125I]hCGRP binding in rat spinal cord in an experimental model of acute, peripheral inflammation.

[125I]Human calcitonin gene-related peptide ([125I]hCGRP) binding in the dorsal horn of the spinal cord exhibited differential changes among laminae over time in response to unilateral adjuvant-induced inflammation. In laminae I/II, 4 days after induction of inflammation, the binding decreased 36% on the side of the spinal cord ipsilateral to the inflammation, while there was no change on the contralateral side. The decrease ipsilateral to inflammation was due primarily to a decrease in the Bmax of the high affinity binding site for CGRP. In lamina V, the binding increased 18% on both sides of the spinal cord at the same time point. In lamina X, the binding increased 16% on both sides of the spinal cord at 2 days after induction of inflammation and remained increased at 8 days. The increases in [125]hCGRP binding in laminae V and X were primarily due to a decrease in the Kd of the low affinity binding site for CGRP. the accompanying hyperalgesia was first measured at 2 days after induction of inflammation and persisted at 8 days. Because the changes in [125I]hCGRP binding did not parallel the hyperalgesia accompanying the unilateral adjuvant-induced inflammation, we believe that CGRP receptors are not directly involved with the hyperalgesia but may be involved with other plastic changes observed in the spinal cord during unilateral adjuvant-induced inflammation.     

Differential Distribution of Thyroid Hormone Receptor Isoform in Rat Dorsal Root Ganglia and Sciatic Nerve in vivo and in vitro

Using autoradiographic techniques carried out under precise conditions we previously demonstrated that both sensory neurons and peripheral glial cells in dorsal root ganglia (DRG) or sciatic nerve, possess specific []> ¹²⁵I]-labeled T₃ binding sites. Thyroid hormone receptors (TR) include several isoforms (TRα₁, TRα₂, TRβ₁, TRβ_2...) The present study demonstrates that while sensory neurons and peripheral glial cells both possess functional TR, they express a differential expression of TR isoforms.     

Neuropeptide receptors in developing and adult rat spinal cord: An in vitro quantitative autoradiography study of calcitonin gene-related peptide,neurokinins, μ-opioid,galanin, somatostatin,neurotensin and vasoactive intestinal polypeptide receptors

A number of neuroactive peptides including calcitonin gene-related peptide (CGRP), substance P, neurokinin B, opioids, somatostatin (SRIF), galanin, neurotensin and vasoactive intestinal polypeptide (VIP) have been localized in adult rat spinal cord and are considered to participate either directly and/or indirectly in the processing of sensory, motor and autonomic functions. Most of these peptides appear early during development, leading to the suggestion that peptides, in addition to their neurotransmitter/neuromodulator roles, may possibly be involved in the normal growth and maturation of the spinal cord. To provide an anatomical substrate for a better understanding of the possible roles of peptides in the ontogenic development of the cord, we investigated the topographical profile as well as variation in densities of [¹²⁵I]hCGRPα, [¹²⁵I]substance P/neurokinin-1 (NK-1), [¹²⁵I]eledoisin/neurokinin-3 (NK-3), [¹²⁵I]FK 33–824 ([D-Ala², Me-Phe⁴, Met(O)ol⁵]enkephalin)/μ-opioid, [¹²⁵I]galanin, [¹²⁵I]T₀D₈-SRIF₁₄ (an analog of sornatostatin), [¹²⁵I]neurotensin and [¹²⁵I]VIP binding sites in postnatal and adult rat spinal cord using in vitro quantitative receptor autoradiography. Receptor binding sites recognized by each radioligand are found to be distributed widely during early stages of postnatal development and then to undergo selective modification to attain their adult profile of distribution during the third week of postnatal development. The apparent density of various receptor sites, however, are differently regulated depending on the lamina and the stage of development studied. For example, the density of μ-opioid binding sites, following a peak at postnatal day 4 (N), declines gradually in almost all regions of the spinal cord with the increasing age of the animal [¹²⁵I]substance P/NK-1 binding sites, on the other hand, show very little variation until P14 and then subsequently decrease as the development proceeds. In the adult rat, most of these peptide receptor binding sites are localized in relatively high amounts in the superficial laminae of the dorsal horn. To varying extents, moderate to low density of various peptide receptor binding sites are also found to be present in the ventral horn, intermediolateral cell column and around the central canal. Taken together, these results suggest that each receptor-ligand system is regulated differently during development and may each uniquely be involved in cellular growth, differentiation and in maturation of the normal neural circuits of the spinal cord, Furthermore, the selective localization of various receptor binding sites in adult rat spinal cord over a wide variety of functionally distinct regions reinforces the neurotransmitter/ modulator roles of these peptides in sensory, motor and autonomic functions associated with the spinal cord. © 1995 Wiley-Liss, Inc.     

Altered Calcitonin Gene-related Peptide, Substance P and Enkephalin Immunoreactivities and Receptor Binding Sites in the Dorsal Spinal Cord of the Polyarthritic Rat

The dorsal horn of the spinal cord, which forms the locus of first synapses in pain pathways, is an important site of interaction between calcitonin gene-related peptide (CGRP), substance P and enkephalin—the neuropeptides considered to be especially involved in the regulation of pain perception. Since adjuvant-induced arthritic rats provide a suitable model for peripheral inflammation and hyperalgesia, the possible alterations of immunoreactive CGRP, substance P and enkephalin as well as the binding sites for [¹²⁵I]hCGRPα, [¹²⁵I]substance Plneurokinin-1, (NK1) and [¹²⁵I]FK-33-824/μ-opioid receptors were studied in the dorsal horn of the spinal cord receiving projections from the inflamed limbs. In arthritic rats compared to control animals, a bilateral increase in CGRP- and substance P-immunoreactive fibres and the presence of enkephalin-immunoreactive cell bodies were noted in the dorsal horn of the spinal cord. As for receptors, while a significant decrease in [¹²⁵I]hCGRPα and [¹²⁵I]substance P/NK1 binding sites was observed in selective layers, no measurable alteration in [¹²⁵I]FK-33-824/μ-opioid binding sites was noted in any regions of the arthritic rat dorsal horn compared to the unaffected control rats. Following unilateral section of the peripheral nerve prior to induction of arthritis, CGRP- and substance P-immunoreactive fibres were markedly depleted and no enkephalin-positive neurons were observed in the ipsilateral dorsal horn. Analysis of receptor binding sites in denervated arthritic rats, however, exhibited differential responses, i.e. a significant increase in [¹²⁵I]hCGRPα, a marked decrease in [¹²⁵I]FK-33-824/μ-opioid and apparently no alteration in [¹²⁵I]substance P/NK1 receptor binding sites were observed in the ipsilateral dorsal horn compared to the intact contralateral side. These results taken together provide anatomical evidence for a concerted role of these peptides in the regulation of adjuvant-induced hyperalgesia accompanying peripheral inflammation.     

A role for bombesin in sensory processing in the spinal cord

Bombesin (BN)-containing neuronal processes were demonstrated in laminae I and II of the dorsal horn of the cat, rat, and mouse spinal cord by immunocytochemistry and radioimmunoassay. Dorsal rhizotomy in the cat resulted in a marked decrease in BN immunoreactivity in the dorsal horn indicating that BN is contained in primary sensory afferents. BN-binding sites were also localized in superficial laminae of the dorsal horn. The presence of both BN and BN-binding sites in the dorsal horn suggested that BN may be involved in sensory processing in the spinal cord. Consistent with this hypothesis, it was demonstrated that an injection of BN into the spinal cord caused a biting and scratching response indicative of sensory stimulation. The effect was similar to that observed after injection of substance P into the cord with the exception that the BN effect lasted about 100 times longer than that induced by substance P. Taken together, these data indicate that BN may be a neurotransmitter of primary sensory afferents to the spinal cord.     

Localization of Dopamine D2 Receptor in Rat Spinal Cord Identified with Immunocytochemistry and In Situ Hybridization

In the present study the distribution of dopamine D₂ receptors in rat spinal cord was determined by means of immunocytochemistry using an anti-peptide antibody, directed against the putative third intracellular loop of the D₂ receptor and in situ hybridization (ISH) using a [³⁵S]UTP labelled anti-sense riboprobe. With the immunocytochemical technique, labelling was confined to neuronal cell bodies and their proximal dendrites. Strongest labelling was present in the parasympathetic area of the sacral cord and in two sexually dimorphic motor nuclei of the lumbosacral cord, the spinal nucleus of the bulbocavernosus and the dorsolateral nucleus. Moderately labelled cells were present in the intermediolateral cell column, the area around the central canal and lamina I of the dorsal horn. Weak labelling was present in the lateral spinal nucleus and laminae VII and VIII of the ventral horn. Except for the two sexually dimorphic motornuclei of the lumbosacral cord labelled motoneurons were not encountered. With the ISH technique radioactive labelling was present in many neurons, indicating that they contained D₂ receptor mRNA. The distribution of these neurons was very similar to the distribution obtained with immunocytochemistry, but with ISH additional labelled cells were detected in laminae III and IV of the dorsal horn, which were never labelled with immunocytochemistry. The present study shows that the D₂ receptor is expressed in specific areas of the rat spinal cord. This distribution provides anatomical support for the involvement of D₂ receptors in modulating nociceptive transmission and autonomic control. Our data further indicate that D₂ receptors are not directly involved in modulating motor functions with the exception, possibly, of some sexual motor functions.     

Spinal release of immunoreactive dynorphin A(1−8) with the development of peripheral inflammation in the rat

Microprobes bearing immobilised antibodies to dynorphin A₍₁₋₈₎ were used to study the basal and evoked release of this prodynorphin derived peptide in the spinal cord of urethane anaesthetised normal rats and those with a peripheral inflammation. In the absence of any active peripheral stimulus the antibody microprobes detected immunoreactive (ir)-dynorphin A₍₁₋₈₎ in two areas (lamina I and laminae IV–V) in the dorsal horn of the spinal cord of normal rats. With the development of unilateral ankle inflammation over 3 to 5 days following subcutaneous injections of Freund's complete adjuvant, a basal presence of ir-dynorphin A₍₁₋₈₎ was found in both the dorsal and ventral horn regions of both sides of the spinal cord. Lateral compression of the ankles of the normal animals did not release ir-dynorphin A₍₁₋₈₎ during the period of stimulation, but this neuropeptide was detected in increased amounts in the ventral horn following the stimulus. By contrast, compression of inflamed ankles produced elevated levels of ir-dynorphin A₍₁₋₈₎ during the period of stimulus application at three major sites in the ipsilateral spinal grey matter. The largest peak was in the deep dorsal horn/upper ventral horn (laminae VI–VII), with further sites of significant release in the mid dorsal horn (laminae II–V) and the lower ventral horn. The observation that ir-dynorphin A₍₁₋₈₎ is physiologically released in the ventral and deep dorsal in addition to the superficial dorsal horn of the rat suggests an involvement of dynorphins in several aspects of spinal function.     

Evidence for Multiple Opioid Receptors in the Human Posterior Pituitary

The distribution and quantification of opioid receptor types in post-mortem human pituitary cryostat sections was determined by quantitative in vitro receptor autoradiography. Highly specific radioligands were used for each opioid receptor type, i.e. [¹²⁵I]-FK-33-824 for μ-opioid sites, [¹²⁵I][D.Ala²]-Deltorphin-I for δ -opioid sites and ³H-U69,593 for κ -opioid sites.
None of the five specimens tested exhibited any labelling in the anterior lobe of the pituitary for the three radioligands. As for the posterior pituitary, all 5 specimens contained both μ and κ -opioid binding sites whereas none of them showed δ -binding sites.
The presence of both μ- and κ -opioid binding sites in the human posterior pituitary contrasts with previous findings in the rat where only κ -sites have been found. The present study could contribute to understanding of the functional action of opioids in the human pituitary.     

Time-related decreases in mu and delta opioid receptors in the superficial dorsal horn of the rat spinal cord following a large unilateral dorsal rhizotomy.

The aim of the present study was to measure the time-related modifications of mu and delta opioid binding sites in the superficial layers of the dorsal horn of the rat spinal cord after a C4-T2 unilateral dorsal rhizotomy. Using specific ligands, namely [3H]DAMGO for mu sites and [3H]DTLET for delta sites, and a quantitative autoradiographic analysis, we have observed: (a) a decrease in binding on the ipsilateral side to the lesion as early as the first day postrhizotomy, the maximal loss being attained at 8 days postlesion, (b) after 8 days postlesion, the residual binding remains stable over the period of analysis (90 days), (c) the loss of mu receptors (71-74%) is significantly more pronounced than the loss of delta receptors (57-62%) and (d) affinities of postsynaptic mu and delta receptors are similar to those of the total receptor population in the superficial layers of the dorsal horn. Comparison of these results with the degeneration of primary afferent fibers reported in literature favors the localization of the majority of mu and delta opioid binding sites on fine diameter primary afferent fibers.     

Somatostatin Depletion by Cysteamine Increases Somatostatin Binding and Growth Hormone-Releasing Factor Messenger Ribonucleic Acid in the Arcuate Nucleus

We have previously described somatostatin (SRIF) pericellular binding sites in the vicinity of growth hormone-releasing factor (GRF)-containing cells in the ventrolateral part of the arcuate nucleus (ARC) of the male rat. To further assess the direct role of SRIF on GRF messenger ribonucleic acid (mRNA) levels in the mediobasal hypothalamus, we depleted endogenous SRIF by cysteamine (CS; 300 mg/kg body wt 6 h prior to sacrifice). In the ventrolateral part of the ARC, there was a 2-fold increase (P<0.05) in [¹²⁵I]SRIF specific binding and GRF mRNA-labelled cell numbers in the CS-treated group as compared to control animals. Furthermore, there was a positive correlation between [¹²⁵I]SRIF binding and the number of GRF mRNA-labelled cells (r = 0.89; P<0.01). In contrast, such effects were not observed along the base of the ventromedial nucleus where pericellular [¹²⁵I]SRIF binding was not associated with GRF mRNA-labelled cells. These results provide functional evidence for a direct SRIF inhibition, through specific receptors, of GRF mRNA levels in ARC neurons.     

Insulin-like growth factor-II/Mannose-6-phosphate receptor in the spinal cord and dorsal root ganglia of the adult rat

The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is a multifunctional transmembrane glycoprotein, which interacts with a number of molecules, including IGF-II and M6P-containing lysosomal enzymes. The receptor is widely distributed throughout the brain and is known to be involved in lysosomal enzyme trafficking, cell growth, internalization and degradation of IGF-II. In the present study, using autoradiographic, Western blotting and immunocytochemical methods, we provide the first report that IGF-II/M6P receptors are discretely distributed at all major segmental levels of the spinal cord and dorsal root ganglia of the adult rat. In the spinal cord, a high density of [(125)I]IGF-II binding sites was evident in the ventral horn (lamina IX) and in areas around the central canal (lamina X), whereas intermediate grey matter and dorsal horn were associated with moderate receptor levels. The dorsal root ganglia exhibited rather high density of [(125)I]IGF-II binding sites. Interestingly, meninges present around the spinal cord displayed highest density of [(125)I]IGF-II binding compared to any given region of the spinal grey matter or the dorsal root ganglia. Western blot results indicated the presence of the IGF-II/M6P receptor at all major levels of spinal cord and dorsal root ganglia, with little segmental variation. At the cellular level, spinal motorneurons demonstrated the most intense IGF-II/M6P receptor immunoreactivity, followed by interneurons in the intermediate region and deeper dorsal horn. Some scattered IGF-II/M6P immunoreactive fibers were found in the superficial laminae of the dorsal horn and dorsolateral funiculus. The meninges of the spinal cord also seemed to express IGF-II receptor immunoreactivity. In the dorsal root ganglia, receptor immunoreactivity was evident primarily in a subset of neurons of all diameters. These results, taken together, provide anatomical evidence of a role for the IGF-II/M6P receptor in general cellular functions such as transport of lysosomal enzymes and/or internalization followed by clearance of IGF-II in the spinal cord and dorsal root ganglia.     

Peripheral nerve injury induces reorganization of galanin-containing afferents in the superficial dorsal horn of monkey spinal cord

Peripheral nerve injury-induced structural and chemical modifications of the sensory circuits in the dorsal horn of the spinal cord contribute to the mechanism of neuropathic pain. In contrast to the topographic projection of primary afferents in laminae I-IV in the rat spinal cord, the primary afferents of Macaca mulatta monkeys almost exclusively project into laminae I-II of the spinal cord. After peripheral nerve injury, up-regulation of galanin has been found in sensory neurons in both monkey and rat dorsal root ganglia. However, the nerve injury-induced ultrastructural modification of galanin-containing afferents in the monkey spinal cord remains unknown. Using immunoelectron microscopy, we found that 3 weeks after unilateral sciatic nerve transection, the number of galanin-containing afferents was increased in ipsilateral lamina II of monkey spinal cord. Branching of these galanin-positive afferents was often observed. The afferent terminals contained a large number of synaptic vesicles, peptidergic vesicles and mitochondria, whereas the number of synapses was markedly reduced. Some of the afferents-enriched microtubules were often packed into bundles. Moreover, galanin-labeling could be associated with endosomal structures in many dendrites and axonal terminals of dorsal horn neurons. These results suggest that peripheral nerve injury induces an expansion of the central projection of galanin-containing afferents in lamina II of the monkey spinal cord, not only by increasing galanin levels in primary afferents but also by triggering afferent branching.     

PACAP mRNA is expressed in rat spinal cord neurons

This study examines the expression of pituitary adenylate cyclase activating polypeptide (PACAP) mRNA in the rat spinal cord during normal conditions and in response to sciatic nerve transection. Previously, PACAP immunoreactivity has been found in fibers in the spinal cord dorsal horn and around the central canal and in neurons in the intermediolateral column (IML). Furthermore, in the dorsal root ganglia, PACAP immunoreactivity and PACAP mRNA expression have been observed preferentially in nerve cell bodies of smaller diameter terminating in the superficial laminae of the dorsal horn. However, neuronal expression of PACAP mRNA in adult rat spinal cord appeared limited to neurons of the IML. By using a refined in situ hybridization protocol, we now detect PACAP mRNA expression in neurons primarily in laminae I and II, but also in deeper laminae of the spinal cord dorsal horn and around the central canal. In addition, PACAP mRNA expression is observed in a few neurons in the ventral horn. PACAP expression in the ventral horn is increased in a population of large neurons, most likely motor neurons, both after distal and proximal sciatic nerve transection. The proposed role of PACAP in nociception is strengthened by our findings of PACAP mRNA-expressing neurons in the superficial laminae of the dorsal horn. Furthermore, increased expression of PACAP in ventral horn neurons, in response to nerve transection, suggests a role for PACAP in repair/regeneration of motor neurons.     

Identified spinal motoneurons of young rats possess nicotinic acetylcholine receptors of the heteromeric family

The aim of the present study was to determine whether, in young rats, spinal motoneurons possess functional nicotinic acetylcholine receptors. Motoneurons were identified either by retrograde labelling or by choline acetyltransferase immunohistochemistry. Whole-cell recordings were performed in spinal cord slices cut at the lumbar level. In voltage clamp, acetylcholine evoked a rapidly activating inward current. In current clamp, it depolarized the motoneuron membrane and induced action potential firing. The acetylcholine-evoked current was strongly reduced by d-tubocurarine or dihydro-beta-erythroidine, broad spectrum nicotinic antagonists, but was almost insensitive to methyllycaconitine, a nicotinic antagonist selective for receptors containing the alpha7 subunit. Moreover, exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane, an alpha7-specific agonist, was without effect. In young animals, light-microscopic autoradiography showed that in the central grey matter all laminae were intensely and equally labelled by [3H]epibatidine. A dense [125I]-alpha-bungarotoxin binding was also found in all laminae, with slightly lower levels in the superficial layers of the dorsal horns and in the ventral part of the grey matter. In adults, the density of [3H]epibatidine binding sites was much lower in the entire grey matter, except in layer 2 of the dorsal horn, and [125I]-alpha-bungarotoxin binding sites were present only in some selected areas. Our data indicate that spinal motoneurons possess functional nicotinic receptors of the heteromeric type and suggest that nicotinic cholinergic transmission may play a significant role in the developing spinal cord.