Subacute administration of a TRH analogue (RX77368) in motorneuron disease: an open study.

Sixteen patients with motor neuron disease received RX77368, a TRH analogue, IV, repeatedly over 1-12 weeks (median 2 weeks). Slight to moderate improvement in bulbar function, particularly speech, was reproduced or persisted with repeated infusions in 8 of 12 responders over a median of 18 days (range 14-90) during the period of study. Cramps (5/9) and spasticity (5/8) improved for a median of 14 days (range 7-35) and 7 days (range 2-14) respectively. The highest benefit/side effect ratio was seen with 0.2 mg/kg (0.15 mg/kg in those with severe bulbar palsy) every 3-4 days. Long term studies with this analogue in MND are indicated.     

Comparative efficacy and safety of intravenous and oral administration of a TRH analogue (RX77368) in motor neuron disease.

Ten consecutive patients with motor neuron disease (MND) who had bulbar symptoms received one or two intravenous doses followed by increasing oral doses of a TRH analogue (RX77368). Similar improvements in speech, swallowing and in tongue and jaw movements were seen after iv and oral administration in nine, five and eight patients respectively. The initial time course of improvement correlated with increasing plasma levels of the drug, but most clinical effects persisted when the levels decreased and became undetectable after 24 hours. The oral solution was tasteless and had no, or minimal, side effects.     

Spinal effects of chronic intrathecal administration of the thyrotrophin-releasing hormone analogue (CG 3509) in rats

The effect of repeated intrathecal administration of a thyrotrophin-releasing hormone (TRH) analogue (CG 3509; 2 μg twice daily for 5 days) on wet-dog shake (WDS) and forepaw-licking (FPL) behaviours and spinal cord TRH and indoleamine levels and choline acetyltransferase (ChAT) activity was examined in adult rats. A rapid behavioural tolerance developed to repeated intrathecal injections of CG 3509; WDS and FPL behaviours were reduced by 57% and 34%, respectively, following the fifth injection and remained reduced at the ninth injection. Repeated CG 3509 administration selectively elevated ChAT activity and the level of 5-hydroxytryptamine (5-HT) in the ventral but not in the dorsal horn of the spinal cord, while 5-hydroxyindoleacetic acid (5-HIAA) and TRH levels were unaltered in either region. As ventral horn ChAT activity is principally located within motoneurones this data implies that TRH exerts a trophic-like influence on mature rat motoneurones in vivo. The results also suggest that long-term intrathecal TRH administration may decrease the release of 5-HT from bulbospinal raphe neurones.     

Evidence for differential storage of calcitonin gene-related peptide, substance P and serotonin in synaptosomal vesicles of rat spinal cord

Homogenates of rat ventral and dorsal spinal cords were subjected to differential and gradient centrifugation on linear sucrose gradients following lysis of a synaptosomal fraction. The distribution of calcitonin gene-related peptide (CGRP) and substance P (SP) in dorsal spinal cord as well as the distribution of serotonin (5-HT) and SP in ventral spinal cord was determined, using radioimmunoassay (RIA) for CGRP and SP and high-performance liquid chromatography for 5-HT. In dorsal spinal cord. CGRP and SP had an almost identical distribution, with one peak in an intermediate density fraction which according to electron microscopy contained i.a. large dense-cored vesicles, and a second peak in a heavy fraction enriched in synaptosome-like structures. In ventral spinal cord, 5-HT and SP had different distribution patterns; 5-HT peaked in a light fraction containing many small synaptic vesicles, whereas SP peaked in an intermediate density fraction similar to the one in dorsal spinal cord. Both 5-HT and SP had second peaks in high density fractions containing synaptosome-like structures. The study demonstrates that the peptides CGRP and SP, partly coexisting in dorsal spinal cord, both seem to be stored in large vesicles inside synaptosomes, whereas the amine 5-HT and the peptide SP, coexisting in ventral spinal cord, appear to have a differential storage with 5-HT mainly in small vesicles and SP in large vesicles, both inside synaptosomes.     

Chronic immunization of endogenous thyrotrophin-releasing hormone (TRH) in brain alters the behavioural response to pentobarbital and a TRH analogue

Rats were infused with purified thyrotrophin-releasing hormone (TRH) anti-serum i.c.v. for two weeks and the reversal of pentobarbital-induced anaesthesia, hypothermia and respiratory depression by central administration of a TRH analogue (CG 3509) was measured. After antibody infusion the anaesthesia time was more than doubled but the responses to CG 3509 were increased, suggesting a role for endogenous TRH in arousal mechanisms which is sensitized following chronic immunological blockade.     

Distribution of a splice variant of choline acetyltransferase in the trigeminal ganglion and brainstem of the rat: comparison with calcitonin gene-related peptide and substance P

Rat trigeminal ganglion neurons have been shown to contain a splice variant of choline acetyltransferase (pChAT). Here we report the distribution pattern of pChAT-containing afferents from the trigeminal ganglion to the brainstem, compared with that of calcitonin gene-related peptide (CGRP) and substance P (SP), by use of the immunohistochemical techniques in the rat. Most of CGRP(+) SP(+) ganglion cells contain pChAT, whereas half of the pChAT(+) ganglion cells possess neither CGRP nor SP. In the brainstem, pChAT(+) nerve fibers are found exclusively in the trigeminal and solitary systems, although the distribution pattern differs from that of CGRP(+) or SP(+) fibers. First, the ventral portion of the principal sensory nucleus contains many pChAT(+) fibers, with few CGRP(+) or SP(+) fibers. Because this portion receives projections of nociceptive corneal afferents, a subpopulation of pChAT(+) CGRP(-) SP(-) primary afferents is most probably nonpeptidergic nociceptors innervating the cornea. Second, the superficial laminae of the medullary dorsal horn, the main target of nociceptive afferents, contain dense CGRP(+) and SP(+) fibers but sparse pChAT(+) fibers. Because pChAT occurs in most CGRP(+) SP(+) ganglion cells, such sparseness of pChAT(+) fibers implies poor transportation of pChAT to axon branchlets. Another important finding is that pChAT(+) axons are smooth and nonvaricose, whereas CGRP(+) or SP(+) fibers possess numerous varicosities. Our confocal microscopy suggests colocalization of these three markers in the same single axons in some brainstem regions. The difference in morphological appearance, nonvaricose or varicose, appears to reflect the difference in intraaxonal distribution between pChAT and CGRP or SP.     

Distribution of secretoneurin immunoreactivity in the spinal cord and lower brainstem in comparison with that of substance P and calcitonin gene-related peptide

Secretoneurin is a peptide of 33 amino acids generated in brain by proteolytic processing of secretogranin II. The distribution of this newly characterized peptide was investigated by means of immunocytochemistry and in situ hybridization in the spinal cord and lower brainstem of the rat. The staining pattern of secretoneurin immunoreactivity (IR) was compared to that of substance P (SP) and calcitonin gene-related peptide (CGRP) in adjacent sections. A high density of secretoneurin-IR fibers and terminals was found in lamina I and outer lamina II of the caudal trigeminal nucleus and of the spinal cord at all levels, around the central canal, and in the sympathetic and parasympathetic areas of the lateral cell columns. The ventral horn displayed a low to moderate density of secretoneurin-IR. The highest number of secretogranin II mRNA-containing cells was found in lamina II of the dorsal horn and in neurons of the dorsal root ganglia. In the white matter, secretoneurin-IR was most prominent in the dorsolateral part of the lateral funiculus and in the tract of Lissauer. The distributions of secretoneurin-IR and SP-IR were strikingly similar. CGRP-IR and secretoneurin-IR overlapped in the outer laminae of the dorsal horn, in the lateral cell column, and probably in some motoneurons. This study establishes that, like SP and CGRP, secretoneurin is a peptide highly concentrated in the terminal field of primary afferents and in sympathetic and parasympathetic areas. Thus secretoneurin might be involved in the modulation of afferent transmission. © Wiley-Liss, Inc.     

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.     

A light and electron microscopic study of calcitonin gene-related peptide in the spinal cord of the rat

The present study localized calcitonin gene-related peptide at the light and electron microscopic levels in the lumbar spinal cord of the rat. One finding was that axons and terminals were labeled in both lamina I and IIo medially but only in lamina I laterally. The functional implications of this innervation pattern are not clear but presumably this anatomic arrangement bears on both dorsoventral and mediolateral patterns of organization of primary afferent input into the dorsal horn. We also found that although the means of labeled myelinated and unmyelinated axon diameters in the tract of Lissauer were different, there was great overlap in these populations. Furthermore, subcellular localizations indicated that immunostaining of calcitonin gene-related peptide was associated primarily with microtubules in axons and cores of large dense-core vesicles in presynaptic terminals. Finally, labeled presynaptic terminals contained relatively few large dense-core vesicles and formed the presynaptic elements of simple axodendritic contacts almost exclusively. These last findings contrast with localizations of calcitonin gene-related peptide in the monkey, which has many more large dense-core vesicles in labeled terminals and in which a much higher proportion of labeled endings form the central parts of glomeruli.     

Marked depletion of dorsal spinal cord substance P and calcitonin gene-related peptide with intact skin flare responses in multiple system atrophy.

In view of the presence of neuropeptides in spinal cord autonomic pathways, their regional concentration was studied in post mortem thoracic cord from four cases of multiple system atrophy with progressive autonomic failure (MSA). A marked depletion was observed of substance P, its related peptide substance K, and of calcitonin gene-related peptide (CGRP), particularly in dorsal regions where peptide-containing sensory fibres terminate. As substance P and CGRP in primary sensory fibres are considered mediators of skin flares in Lewis' triple response, histamine-induced skin flares were measured in 12 MSA patients and were found to be preserved. These results provide a new key to the classification and aetiology of autonomic and multiple system degenerations, as well as a model to study the role of sensory neuropeptides in man.     

Altered levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity of cat lumbar motoneurons after chronic spinal cord transection

In cats subjected to total spinal cord transection at the lower thoracic level, the calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) was studied in motoneuron cell bodies in the L6-L7 segments. In transected animals, the CGRP-immunoreactive labeling of the motoneurons was virtually absent. When combining the spinal cord transection with a unilateral rhizotomy of all dorsal roots below the transection, however, an apparently normal labelling pattern of CGRP-LI of the motoneurons was displayed on both sides. Thus, surgical interventions which affect afferent pathways to the motoneurons may have influence on the levels of CGRP-LI in otherwise intact motoneurons.     

Expression of calcitonin gene-related peptide in anterior and posterior horns of the spinal cord after brachial plexus injury

This study shows the expression pattern of calcitonin gene-related peptide (CGRP) in the anterior and posterior horns of the spinal cord after brachial plexus injury. The animals were divided into three injury groups: group 1, right C₇ anterior root avulsion; group 2, right C₇ anterior root avulsion and cut right C₅–T₁ posterior roots; and group 3, right C₇ anterior root avulsion plus right hemitransection between the C₅ and C₆ segments of the spinal cord. These animals were killed at 1, 3, 7 and 14 days after injury. In the anterior horn of all three injured groups, the expression of CGRP increased progressively from day 1 to day 7 (p < 0.05), peaked on day 7, and then began to decrease slowly. In the posterior horn of all three injured groups, the expression of CGRP decreased gradually from day 1 to day 14 after the operation and was significantly lower on day 14 compared to day 1. At each time point (days 1, 3, 7 and 14), the expression of CGRP was the highest in group 1 and the lowest in group 2, with significant differences among the three groups. The CGRP in the anterior horn of the spinal cord was derived from the cell bodies of motor neurons and was possibly involved in repair mechanisms and regeneration after nerve injury. However, the CGRP in the posterior horn was mainly derived from the posterior root ganglion and was possibly associated with the conduction of noxious stimulation.     

Crossed and uncrossed projections to the cat sacrocaudal spinal cord: III. Axons expressing calcitonin gene-related peptide immunoreactivity

We have investigated the projection patterns of peptidergic small-diameter primary afferent fibers to the cat sacrocaudal spinal cord, a region associated with midline structures of the lower urogenital system and of the tail. Calcitonin gene-related peptide (CGRP)-immunoreactive (CGRP-IR) primary afferent fibers were observed within the superficial laminae, rostrally as the typical inverted U-shaped band that capped the separate dorsal horns (S1 to rostral S2) and caudally as a broad band that spanned the entire mediolateral extent of the fused dorsal horns (caudal S2 and caudal). Within the dorsal gray commissure, labeling was seen as a periodic vertical, midline band. CGRP-IR labeling was prevalent in an extensive mediolateral distribution at the base of the dorsal horn, originating from both lateral and medial collateral bundles that extend from the superficial dorsal horn. Some bundles, in part traveling within the dorsal commissure, conspicuously crossed the midline. In addition to the robust projection to the superficial dorsal horn, there was a more extensive distribution of CGRP-IR fibers within the deeper portions of the cat sacrocaudal dorsal horn than has been reported for other regions of the cat spinal cord. Presumably, these deep projections convey visceral information to projection or segmental neurons at the neck of the dorsal horn and in the region of the central canal. This deep distribution overlaps the reported projections of the pelvic and pudendal nerves. In addition, the contralateral projections of CGRP-IR fibers may form an anatomical substrate of the bilateral receptive fields for selective dorsal horn neurons. The density and variety of CGRP-IR projection patterns is a reflection of the functional attributes of the innervated structures.     

Distribution of thyrotropin-releasing hormone (TRH) immunoreactivity in the thoracic spinal cord of guinea pig

The localization of thyrotropin-releasing hormone immunoreactivity (TRH-IR) has been determined in the thoracic spinal cord of male and female guinea pigs. The immunoreactive product is localized in nerve fibre varicosites and terminals throughout the spinal gray matter and in some regions of the white matter. There are not TRH-IR neurons in the spinal cord. The highest density of IR structures is observed in the intermediate zone, in the central gray and in the ventral horns, around the motoneurons. Less TRH-IR structures are observed in the superficial layers and substantia gelatinosa of the dorsal horns. Between the ependymal cells of the central canal are observed single TRH-IR fibres and terminals too. Most of the TRH-IR fibres and terminals in the intermediate zone and in the central gray are constituents of the vegetative network (Galabov and Davidoff, 1976 and Davidoff et al. 1985). As to the origin of the spinal cord TRH-IR fibres and terminals two main possibilities exist: a) From primary afferent neurons situated in the dorsal root ganglia, which is quite uncertain; b) From supraspinal neurons which send their axons descending in the white matter and in the fasciculi longitudinales laterales and mediales (Johansson et al. 1981, 1983). The wide diversity of neuroactive substances in the thoracic spinal cord vegetative network and the origin of its fibres and terminals suggests that this network may play an important role in the integration of the central and peripheral vegetative nervous system, as well as probably in the integration of the somatic and the vegetative nervous system.     

Central projection of calcitonin gene-related peptide (CGRP)- and substance P (SP)-immunoreactive trigeminal primary neurons in the rat

Substance P (SP) is implicated in transmission of primary afferent nociceptive signals. In primary neurons, SP is colocalized with calcitonin gene-related peptide (CGRP), which is another neuropeptide marker for small to medium primary neurons. CGRP coreleased with SP augments the postsynaptic effect of SP and thereby modulates the nociceptive transmission. This study demonstrates the distribution of CGRP-like immunoreactivity (-ir) and SP-ir in the lower brainstem of normal rats and after trigeminal rhizotomy or tractotomy at the level of subnucleus interpolaris (Vi). By comparing the results obtained from normal and deafferented rats, we analyzed the central projection of trigeminal primary nociceptors. The CGRP-immunoreactive (-ir) trigeminal primaries projected to the entire rostrocaudal extent of the spinal trigeminal nucleus, the principal nucleus (PrV), the paratrigeminal nucleus (paraV), and the lateral subnucleus of solitary tract nucleus (STN) on the ipsilateral side. The trigeminal primaries projecting to the spinal trigeminal nucleus, paraV and STN also contained SP-ir. The ipsilateral trigeminal primaries were the exclusive source of CGRP-ir terminals in the PrV, the Vi and the dorsomedial nucleus within the subnucleus oralis (Vo). The medullary dorsal horn (MDH) and the lateral edge of Vo received convergent CGRP-ir projection from the ipsilateral trigeminal primaries and other neurons. The glossopharyngeal and vagal primaries are candidates for the source of CGRP-ir projection to the Vo and the MDH, while the dorsal root axons supply the MDH with CGRP-ir terminals. In addition, contralateral primary neurons crossing the midline appear to contain CGRP and to terminate in the MDH. J. Comp. Neurol. 378:425–442, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Calcitonin gene-related peptide (CGRP) in the human spinal cord: a light and electron microscopic analysis

The distribution of CGRP immunoreactivity in the cervical, thoracic, lumbar, and sacral levels of the human spinal cord was mapped at the light microscopic level with the aid of a rabbit-generated antiserum against human calcitonin gene-related peptide (CGRP). CGRP-positive fibers formed a dense plexus in lamina I, II, the reticulated region of lamina V, and the tract of Lissauer at all spinal cord levels. The distribution of fibers showed some variations dependent on the cord level analyzed. At the light microscopic level, intervaricose fiber diameters consistently measured 1.0 micron or less, and two types of CGRP varicosities were observed: a small (1 to 2 microns in diameter), relatively round profile and a larger, (3 to 4 microns in diameter) oval or oblong profile. At the electron microscopic level, immunostained varicosities contained a mixture of round clear vesicles and vesicles that contained dense cores. The CGRP immunoreaction product was often associated with vesicles containing dense cores. The reaction product was also seen associated with clear round vesicles or in the cytoplasmic matrix. Postsynaptic elements included dendritic spines, small and large diameter dendritic shafts and vesicle containing profiles. The presence of CGRP in the superficial dorsal horn of human spinal cord is highly suggestive of a role in primary afferent transmission as postulated in lower vertebrates. This study establishes the distribution of CGRP at four different spinal levels in human cord and will serve as a basis for future studies related to the pathologic conditions affecting sensory systems.     

Presence of calcitonin gene-related peptide (CGRP) and substance P (SP) immunoreactivity in intraepidermal free nerve endings of cat skin

Dorsal roots are absent from the tails of Xenopus larvae. Sensory afferents instead enter the spinal cord via the ventral roots. After reaching the cord these axons travel diagonally within the lateral fasciculus of the cord to reach the dorsolateral fiber bundle in which they ascend to the hindbrain. Sensory afferents entering together in the same ventral root make this traverse individually rather than as a group, but coalesce again at the ascending tract.