Distribution and ultrastructure of tachykinin-like immunoreactivity in the frog (Rana esculenta) spinal cord, notably, the dorsal horn

Tachykinins are involved in pain transmission at the spinal level. In frog, at least four tachykinins [TK] have been isolated from the brain, but their organization in the dorsal horn of the spinal cord is still poorly known. We have reexamined TK distribution by immunocytochemistry using an antibody recognizing the sequence common to all tachykinins in the spinal cord and dorsal root ganglia of the green frog Rana esculenta. A dense tachykinin-like immunoreactivity (TK-LI) was observed in the dorsolateral fasciculus or Lissauer's tract running ventromedial to the entry of the dorsal root and in numerous small and medium-sized dorsal root ganglion cells showing a primary afferent origin for part of TK-LI of the dorsal horn. The observation of numerous cell bodies in the dorsal horn, in addition, suggested a local or propriospinal origin. One group of cells was localized at the entrance of the Lissauer's tract TK-LI fibers into the dorsal horn, and another group was localized in the upper dorsal horn, a region with a low density of TK-LI fibers. It was suggested that the latter group may correspond to neurokinin B. Electron microscopic examination of the Lissauer's tract showed numerous immunoreactive axons, some located at the center of glomerular-like arrangements, suggesting that the information brought by these fibers may be transmitted and most probably modulated before their entry in the dorsal horn. In conclusion, the functional organization of tachykinins in the frog spinal cord seems to be similar to that of mammals, albeit with a different morphological organization.     

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.     

Immunocytochemical localization of neurokinin B in the rat spinal dorsal horn and its association with substance P and GABA: an electron microscopic study

Substance P and neurokinin B are tachykinins that derive from different precursors. Both tachykinins are known to be involved in the processing of pain-related information. Initial studies suggested an antinociceptive effect for neurokinin B, but more recent data indicate that neurokinin B facilitates nociception. Unfortunately, morphologic correlates are lacking, as little is known about the distribution of neurokinin B, especially at the ultrastructural level. Because of its potentially important role in the processing of pain-related information, we decided to investigate the synaptic interactions of neurokinin B-immunoreactive profiles in laminae I-III of the rat cervical spinal dorsal horn and their relation to substance P-immunoreactive structures. An antibody raised against a portion of the neurokinin B precursor peptide was used for the detection of neurokinin B. Neurokinin B-like immunoreactivity occurred in all superficial laminae, with the highest density in inner lamina II and the lowest in lamina III. Neurokinin B-like immunoreactive axonal boutons were mainly dome-shaped and established symmetric synaptic contacts with dendrites or cell bodies. Neurokinin B-like immunoreactivity was also detected in dendritic profiles in all superficial laminae. Some of these dendritic profiles were part of synaptic glomeruli in inner lamina II and lamina III. Double-labeling for neurokinin B and substance P showed a lack of appositions and synapses between neurokinin B and substance P-positive profiles. Furthermore, very few profiles double-labeled for the two peptides were observed. Double-labeling for gamma-aminobutyric acid (GABA) and neurokinin B showed a complete absence of neurokinin B/GABA co-localization. Furthermore, neurokinin B-positive profiles were never presynaptic to GABA-immunoreactive profiles, but frequently neurokinin B-positive dendrites were postsynaptic to GABA-immunoreactive boutons. These results suggest that neurokinin B participates in circuits separate from those involving substance P, as virtually no anatomic correlation was found between the two neuropeptides.     

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.     

Capsaicin depletes CCK-like immunoreactivity detected by immunohistochemistry, but not that measured by radioimmunoassay in rat dorsal spinal cord

In rats treated neonatally with capsaicin there were significant reductions in substance P measured by radioimmunoassay in dorsal spinal cord, spinal ganglia, and coelic ganglia, but concentrations of cholecystokinin octapeptide (CCK8) measured by radioimmunoassay were not decreased. However, immunohistochemical studies using antisera to both substance P and CCK8, respectively, demonstrated decreased immunoreactive material in the dorsal horn of the spinal cord. The nature of the material localized by CCK8 antiserum in immunohistochemistry remains to be resolved.     

Peripheral nerve injury induces trans-synaptic modification of channels, receptors and signal pathways in rat dorsal spinal cord

Peripheral tissue injury-induced central sensitization may result from the altered biochemical properties of spinal dorsal horn. However, peripheral nerve injury-induced modification of genes in the dorsal horn remains largely unknown. Here we identified strong changes of 14 channels, 25 receptors and 42 signal transduction related molecules in Sprague-Dawley rat dorsal spinal cord 14 days after peripheral axotomy by cDNA microarray. Twenty-nine genes were further confirmed by semiquantitative RT-PCR, Northern blotting, in situ hybridization and immunohistochemistry. These regulated genes included Ca2+ channel alpha1E and alpha2/delta1 subunits, alpha subunits for Na+ channel 1 and 6, Na+ channel beta subunit, AMAP receptor GluR3 and 4, GABAA receptor alpha5 subunit, nicotinic acetylcholine receptor alpha5 and beta2 subunits, PKC alpha, betaI and delta isozymes, JNK1-3, ERK2-3, p38 MAPK and BatK and Lyn tyrosine-protein kinases, indicating that several signal transduction pathways were activated in dorsal spinal cord by peripheral nerve injury. These results demonstrate that peripheral nerve injury causes phenotypic changes in spinal dorsal horn. Increases in Ca2+ channel alpha2/delta1 subunit, GABAA receptor alpha5 subunit, Na+ channels and nicotinic acetylcholine receptors in both dorsal spinal cord and dorsal root ganglia indicate their potential roles in neuropathic pain control.     

Immunocytochemical localization of somatostatin receptor sst2A in the rat spinal cord and dorsal root ganglia

Intrathecal administration of octreotide, a stable somatostatin analogue, provides pain relief in patients, and locally applied somatostatin inhibits firing of nociceptive dorsal horn neurons. In the present study, we have raised polyclonal antibodies that specifically detect the somatostatin receptor sst_2A and used these antisera for immunocytochemical localization of the receptor protein in the rat spinal cord and dorsal root ganglia. In the superficial layers of the dorsal horn, sst_2A-like immunoreactivity (Li) formed a dense network consisting of neuronal perikarya and dendrites which were often closely apposed by, but not co-contained within, somatostatin-14-immunoreactive nerve fibres and terminals. sst_2A-Li was resistant to dorsal rhizotomy and did not colocalize with either substance P or calcitonin gene-related peptide suggesting that sst_2A-Li was not located to primary afferents, but rather confined to second-order spinal neurons. The position of sst_2A-Li perikarya and dendrites in the dorsal horn appeared to be similar to those containing μ-opioid receptor-Li; however, double labelling experiments revealed no instances of coexistence of these two receptors. sst_2A-Li was also observed in the dorsal root ganglia predominantly targeted to the somatic plasmalemma of medium size neurons distinct from those expressing somatostatin-14 or δ-opioid receptors. Thus, the present results not only provide a morphological substrate for spinal octreotide analgesia but also show that somatostatin and opioids are poised to modulate nociceptive transmission by distinct anatomical systems.     

Calcium-binding proteins, parvalbumin- and calbindin-D 28k-immunoreactive neurons in the rat spinal cord and dorsal root ganglia: a light and electron microscopic study

The distribution of two calcium-binding proteins, parvalbumin (PV) and calbindin-D 28K (CaBP), was studied by the peroxidase-anti-peroxidase immunohistochemical method at the light and electron microscopic level in the rat spinal cord and dorsal root ganglia. The possible coexistence of these two proteins was also investigated. PV-positive neurons were revealed in all layers of the spinal cord, except lamina I, which was devoid of labelling. Most of the PV-positive cells were found in the inner layer of lamina II, lamina III, internal basilar nucleus, central gray region, and at the dorsomedial and ventromedial aspects of the lateral motor column in the ventral horn. Neuronal processes intensely stained for PV sharply delineated inner lamina II. With the electron microscope most of them appeared to be dendrites, but vesicle containing profiles were also found in a smaller number. CaBP-positive neurons appeared to be dispersed all over the spinal gray matter. The great majority of them were found in laminae I, II, IV; the central gray region; the intermediolateral nucleus; and in the ventral horn just medial to the lateral motor column. Laminae I and II were densely packed with CaBP-positive punctate profiles that proved to be dendrites and axons in the electron microscope. A portion of labelled neurons in lamina IV and on the ventromedial aspect of the lateral motor column in the ventral horn disclosed both PV- and CaBP-immunoreactivity. All of the funiculi of the spinal white matter contained a large number of fibres immunopositive for both PV and CaBP. The highest density of CaBP-positive fibres was found in the dorsolateral funiculus, which was also densely packed with PV-positive fibres. PV-positive fibres were even more numerous in the dorsal part of the dorsal funiculus. The territory of the gracile funiculus in the brachial cord and that of the pyramidal tract in its whole extent were devoid of labelled fibres. In the thoracic cord, the dorsal nucleus of Clarke received a large number of PV-positive fibres. Dorsal root ganglia displayed both PV- and CaBP-immunopositivity. The cell diameter distribution histogram of PV-positive neurons disclosed two peaks--one at 35 microns and the other at 50 microns. CaBP-positive cells in the dorsal root ganglia corresponded to subgroups of small and large neurons with mean diameters of 25 microns and 45 microns, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Substance P-like immunoreactivity in neurons in dissociated cell cultures of mammalian spinal cord and dorsal root ganglia

Dissociated cell cultures prepared from fetal mouse spinal cords and dorsal root ganglia were stained for endogenous substance P using the peroxidase-antiperoxidase technique. Substance P-like immunoreactivity was localized within a small percentage of rounded or multipolar neuronal somata and in varicose processes. The substance P-positive multipolar neurons were derived from spinal cord, while the small rounded neurons were possibly of spinal cord and/or sensory ganglion origin. Large dorsal root ganglion neurons were unreactive. These results are consistent with in vivo findings and indicate the feasibility of electrophysiologic studies in culture to analyze the synaptic connections between substance P neurons and their target cells.     

Increased calcitonin gene-related peptide (CGRP), substance P,and enkephalin immunoreactivities in dorsal spinal cord and loss of CGRP-immunoreactive motoneurons in arthritic rats depend on intact peripheral nerve supply

The distribution of peptides thought to be involved in pain modulation—substance P, calcitonin gene-related peptide (CGRP), and enkephalin—were studied in the spinal cord and dorsal root ganglia of polyarthritic rats and in rats with one sciatic nerve sectioned prior to induction of arthritis. In arthritic rats there was a bilateral increase of CGRP- and substance P-immunoreactive fibers and appearance of enkephalin-immunoreactive cell bodies in the dorsal horn of the lumbar (L₄) spinal cord when compared to controls. In the corresponding dorsal root ganglia there were significant increases of CGRP- (P<0.02) and substance P- (P<0.001) immunoreactive cell bodies compared to controls. In the ventral horn of the control rats CGRP-immunoreactive motoneurons were abundant but were significantly (P<0.001) reduced in the arthritic spinal cord. Less pronounced changes were seen in the contralateral L₄ spinal cord of arthritic rats with unilateral sciatic nerve section. In the ipsilateral dorsal horn, however, CGRP- and substance P-immunoreactive fibers were markedly depleted, and no enkephalin cell bodies were present. Furthermore, a number of CGRP-immunoreactive motoneurons were observed. In the ipsilateral L₄ ganglia CGRP- (P<0.02) and substance P- (P<0.02) immunoreactive cells were significantly decreased compared to the contralateral side. The data suggest that pain perception is linked to complex interactions between CGRP, substance P, and enkephalin in sensory pathways and an intact peripheral input. The loss of CGRP-immunoreactive motoneurons may reflect muscular dysfunction associated with the arthritic condition.     

Functional connections are established in the deafferented rat spinal cord by peripherally transplanted human embryonic sensory neurons

Functionally useful repair of the mature spinal cord following injury requires axon growth and the re-establishment of specific synaptic connections. We have shown previously that axons from peripherally grafted human embryonic dorsal root ganglion cells grow for long distances in adult host rat dorsal roots, traverse the interface between the peripheral and central nervous system, and enter the spinal cord to arborize in the dorsal horn. Here we show that these transplants mediate synaptic activity in the host spinal cord. Dorsal root ganglia from human embryonic donors were transplanted in place of native adult rat ganglia. Two to three months after transplantation the recipient rats were examined anatomically and physiologically. Human fibres labelled with a human-specific axon marker were distributed in superficial as well as deep laminae of the recipient rat spinal cord. About 36% of the grafted neurons were double labelled following injections of the fluorescent tracers MiniRuby into the sciatic and Fluoro-Gold into the lower lumbar spinal cord, indicating that some of the grafted neurons had grown processes into the spinal cord as well as towards the denervated peripheral targets. Electrophysiological recordings demonstrated that the transplanted human dorsal roots conducted impulses that evoked postsynaptic activity in dorsal horn neurons and polysynaptic reflexes in ipsilateral ventral roots. The time course of the synaptic activation indicated that the human fibres were non-myelinated or thinly myelinated. Our findings show that growing human sensory nerve fibres which enter the adult deafferentated rat spinal cord become anatomically and physiologically integrated into functional spinal circuits.     

Immunohistochemical evidence for the selective involvement of dorsal root fibres in Friedreich''s ataxia

An immunohistochemical study was carried out in order to elucidate the selective involvement of the dorsal root fibres from two patients with Friedreich's ataxia in comparison with those of 10 neurologically normal control individuals. For this purpose, antibodies to substance P and to synaptophysin were used. Substance P-immunoreactive unmyelinated fibres forming a dense network in the normal substantia gelatinosa of the spinal dorsal horn predominantly originate from a subpopulation of small cells of the dorsal root ganglia, while synaptophysin is present in virtually all nerve cell axon terminals and is useful for visualizing axon terminals in the nervous system. Strong substance P-like immunoreactivity was seen in the substantia gelatinosa of patients with Friedreich's ataxia. By contrast, there was marked depletion of synaptophysin immunoreactivity in the posterior column nuclei, with the gracile nucleus showing greater loss of positive puncta than the cuneate nucleus.     

The involvement of neurokinin receptor subtypes in somatosensory processing in the superficial dorsal horn of the cat.

As well as substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) have recently been found in the superficial dorsal horn of the spinal cord; NKA originating mainly in fine primary afferents. We have investigated the effects of these tachykinins and a range of analogues on somatosensory responses of single identified dorsal horn neurons, when applied ionophoretically to the region of the substantia gelatinosa. Behavioural reflex tests of thermal nociception were carried out in parallel. The role of NK-1, NK-2 and NK-3 receptors was addressed. NK-1-selective agonists attenuated the non-nociceptive responses of identified multireceptive spinocervical tract (SCT) neurons. Of the endogenous tachykinins, both SP and NKB (a weak NK-1 agonist) showed this effect. No role for NK-3 receptors was identified in our experiments. NK-2-selective agonists (including NKA) caused a unique and selective facilitation of thermal nociceptive responses. NKA also reduced reflex response latency in tail-flick and hot plate tests. NKA as a primary afferent transmitter may thus be involved in mediating or facilitating the expression of thermal nociceptive inputs in the substantia gelatinosa. NKA and SP could be considered as acting in concert in the superficial dorsal horn in an effectively pro-nociceptive modulatory role. Evidence from receptor-selective antagonists supports that obtained with agonists for the roles of particular NK receptors in somatosensory processing. NK-2, but not NK-1 or NK-3 antagonists attenuated endogenous thermal nociceptive responses, supporting the hypothesis that an NK-2 agonist (such as NKA) may normally participate in expression of thermal nociception in the superficial dorsal horn. Behavioural experiments showing increased response latencies with a putative NK-2 selective antagonist further supported the involvement of NK-2 receptors in thermal nociception.     

Changes of chemoarchitectural organization of the rat spinal cord following ventral and dorsal root transection.

Time-related changes in the distribution of chemical messengers in the rat spinal cord following the transection of dorsal and ventral roots were observed by using immunohistochemistry for the following antigens: microtubule-associated protein 2 (MAP2), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (Gal), Met-enkephalin (Enk), neuropeptide Y (NPY), and serotonin (5-HT). To investigate dendrocytoarchitectonic organizational changes, morphometric analyses were performed on both the gray and the white matter of tissue samples stained with MAP2 antiserum. A significant reduction in the area of gray matter on the lesioned side was seen from 1 to 24 weeks postoperation, and progressive changes in the shape of the gray matter were also observed. CGRP-immunoreactive fibers were reduced in number in the posterior horn after root transection, except in the lateral part of lamina I. In contrast, CGRP immunoreactivity in the anterior horn cells of the ipsilateral side was increased early after transection, but later it progressively decreased. Root transection also caused significant reduction in the number of SP-immunoreactive fibers in the posterior horn, but no changes were seen in the anterior horn. Gal immunoreactivity was also affected by root transection, and it changed in a similar way to CGRP immunoreactivity. 5-HT-immunoreactive fibers were increased in the posterior horn after transection, and later decreased. In the anterior horn, there were no changes in the intensity or distribution pattern of 5-HT-immunoreactive nerve fibers following root transection. Enk and NPY immunoreactivity in the anterior and posterior horns was not affected by root transection up to 24 weeks postoperative. These results show that spinal root transection caused significant changes in the chemoarchitectural organization of nerve fibers containing certain types of chemical messengers, such as CGRP, SP, Gal, and 5-HT, in addition to altering dendritic geometry in the spinal cord.     

Ontogeny of peptide- and amine-containing neurones in motor, sensory, and autonomic regions of rat and human spinal cord, dorsal root ganglia, and rat skin

The developmental patterns of neurofilament triplet proteins, peptide and amine immunoreactivities were compared in motor (ventral spinal cord), sensory (dorsal spinal cord, dorsal root ganglia, epidermis), and autonomic (intermediolateral cell columns, dermis) regions in the rat and human. In the rat, neurofilament triplet proteins first appeared in motoneurones (embryonic day 13). In the youngest human fetuses studied (6 weeks), immunoreactivity was present throughout the spinal cord. Peptides and amines occurred later. Calcitonin gene-related peptide, galanin, somatostatin, neuropeptide Y and its C-flanking peptide (CPON) were the first to appear localized to motoneurones (embryonic days 15-17 rat; fetal weeks 6-14 human). Numbers of immunoreactive motoneurones decreased toward birth, but immunoreactive fibers increased in the ventral horn with enkephalin, thyrotrophin-releasing hormone, and the monoaminergic markers 5-hydroxytryptamine and tyrosine hydroxylase (all presumably of supraspinal origin) the last to appear perinatally. In the dorsal horn, particularly in the rat, a transient expression of substance P-, somatostatin-, and neuropeptide Y/CPON-immunoreactive cells was detected (embryonic days 15-17). A pronounced increase of calcitonin gene-related peptide-, galanin-, somatostatin- and substance P- immunoreactive fibers was found perinatally in both species. This coincided with an increased detection of cells in the dorsal root ganglia containing these peptides and the earliest appearance of calcitonin gene-related peptide-, somatostatin-, and substance P-immunoreactive fibers in the rat epidermis. Few antigens were localized to the intermediolateral cell columns before embryonic day 20 (rat), fetal week 20 (human), with thyrotrophin-releasing hormone-, 5-hydroxytryptamine-, tyrosine hydroxylase-, and vasoactive intestinal polypeptide-immunoreactive nerves appearing perinatally. In the rat dermis, tyrosine hydroxylase-immunoreactive fibers (sympathetic fibers) and fibers immunoreactive for neuropeptide Y/CPON and vasoactive intestinal polypeptide were detected from postnatal day 1. In conclusion, 1) peptide and amine immunoreactivity develops in motor before sensory or autonomic regions, 2) many peptide-containing cells are transient in fetal life, and 3) central terminals of dorsal root ganglion cells express peptides before terminals in the skin.     

Substance P: Depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurons

The substance P content, glutamic acid decarboxylase and choline acetyltransferase activities and the level of [3H]diprenorphine binding were measured in various regions of the lumbar spinal cord of rats after unilateral section of the sciatic nerve or after dorsal rhizotomy. Sciatic nerve section produced a 75--80% depletion of substance P in the dorsal horn but did not change the substance P content of the ventral horn. The onset of substance P depletion occurred within 7 days and was maintained for 2 months. The substance P content of the dorsal root ganglia and both the peripheral and central branches of primary sensory neurons was also reduced after sciatic nerve section. Glutamic acid decarboxylase and choline acetyltransferase activity were unchanged; however, a small decrease in opiate receptor binding occurred 1 month after nerve section. Dorsal rhizotomy produced an 80% depletion of substance P in the dorsal horn. In addition, the substance P content of the ventral horn was significantly reduced. Glutamic acid decarboxylase activity in the dorsal horn was unaffected by dorsal rhizotomy whereas opiate receptor binding was reduced by 40%. From these studies it appears that peripheral nerve injury results in the degeneration of primary sensory neurons which contain and release substance P as neurotransmitter.     

Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species

Calcitonin gene-related peptide (CGRP) immunoreactivity was found throughout the entire spinal cord of man, marmoset, horse, pig, cat, guinea pig, mouse, rat, and frog. CGRP-immunoreactive fibers were most concentrated in the dorsal horn. In the ventral horn of some species large immunoreactive cells, tentatively characterized as motoneurons, were present. Pretreatment of rats with colchicine enhanced staining of these large cells but did not reveal CGRP-immunoreactive cell bodies in the dorsal horn. In the dorsal root ganglia, CGRP immunoreactivity was observed in most of the small and some of the intermediate sized cells. Substance P immunoreactivity, where present, was co-localized with CGRP to a proportion of the small cells. In the cat the ratio of substance P-immunoreactive to CGRP-immunoreactive ganglion cells was 1:2.7 (p less than 0.001). The concentration of CGRP-immunoreactive material in tissue extracts was determined by radioimmunoassay. In the dorsal horn of the rat spinal cord the levels of peptide were found to range from 225.7 +/- 30.0 pmol/gm of wet weight in the cervical region to 340.6 +/- 74.6 pmol/gm in the sacral spinal cord. In the rat ventral spinal cord, levels of 15.7 +/- 2.7 to 35.1 +/- 10.6 pmol/gm were found. The concentration in dorsal root ganglia of the lumbar region was 225.4 +/- 46.9 pmol/gm. Gel permeation chromatography of this extractable CGRP-like immunoreactivity revealed three distinct immunoreactive peaks, one eluting at the position of synthetic CGRP and the others, of smaller size, eluting later. In cats and rats, rhizotomy induced a marked loss of CGRP-immunoreactive fibers from the dorsal horn of the spinal cord. In the cat, unilateral lumbosacral dorsal rhizotomy resulted in a significant (p less than 0.05) reduction of extractable CGRP from the ipsilateral lumbar dorsal horn (5.6 +/- 1.2 pmol/gm of wet weight) compared to the contralateral side (105.0 +/- 36.0 pmol/gm of wet weight). We conclude that the major origin of CGRP in the dorsal spinal cord is extrinsic, from afferent fibers which are probably derived from cells in the dorsal root ganglia. The selective distribution of CGRP throughout sensory, motor, and autonomic areas of the spinal cord suggests many putative roles for this novel peptide.     

Characterization of forms of immunoreactive somatostatin in sensory neuron and normal and deafferented spinal cord

In order to determine the contribution made by primary sensory afferents and supraspinal projections to the immunoreactive somatostatin (IRS) content of the spinal cord, measurements were made of the concentration of IRS in the dorsal and ventral halves of the cord in cats subjected to unilateral lumbosacral dorsal rhizotomy (L1-S3) alone or combined with spinal cord transection. The molecular forms of IRS (characterized by gel chromatography) in L7 lumbar spinal cord, L6-S1 dorsal roots, ventral roots and dorsal root ganglia, and sciatic nerve were also determined. S14 was the predominant form in all tissues examined, but two additional molecular forms corresponding to S28 and S11.5 kdalton were present in dorsal root ganglia and spinal cord; S28 but not S11.5 kdalton was detected in both dorsal roots and sciatic nerves. These results indicate that S14 and S28 and S28 are transported along the central and peripheral processes of dorsal root ganglia, but that spinal cord S11.5 kdalton originates in the central nervous system. IRS in the dorsal horn was reduced by ca. 40% following dorsal root section. Neither disruption of descending pathways by spinal transection nor surgical isolation of the lumbar segements lowered cord somatostatin content below that produced by dorsal root section, indicating that most of the somatostatin within the cord arises from the dorsal root and from neurons in local spinal segments. Although the total content of IRS in the dorsal horn was reduced by ca. 40% following dorsal rhizotomy, the pattern of molecular forms was not changed accordingly. Since S14 and S28 but not S11.5 kdalton are transported via the dorsal root, the dorsal root section would be predicted to produce a relatively greater decrease in S14 and S28 than in S11.5 kdalton. Therefore, failure to find a selective loss of S14 and S28 suggests that dorsal rhizotomy affects dorsal horn IRS content not only by removing afferent input but possibly also by modifyinh the processing of IRS by the remaining somatostatinergic neurons.