Quinolinic and kainic acids can enhance calcitonin gene-related peptide-like immunoreactivity in striatal neurons with substance P-like immunoreactivity

Quinolinic or kainic acid, when injected into the cat striatum, caused neurodegenerative changes in striatal neurons. In parallel with these changes, however, calcitonin gene-related peptide-like immunoreactivity became visible in perikarya of many striatal neurons which were distributed around the lesions produced by the quinolinic or kainic acid injections. The vast majority of these lesion-spared neurons showing calcitonin gene-related peptide-like immunoreactivity were striatal neurons with substance P-like immunoreactivity.     

Preganglionic and sensory axons in developing bullfrog sympathetic ganglia express three neuropeptides during early tadpole stages.

Retrograde tracing in fixed tissue with the fluorescent carbocyanine dye, DiI, was used to identify neurons that project to paravertebral sympathetic ganglia 9 and 10 in bullfrog tadpoles. Applying DiI to ganglion 9 at stage II labelled spinal preganglionic neurons and sensory neurons in dorsal root ganglia. When examined in a stage V tadpole, the segmental boundaries of the preganglionic cell column which supply the lumbar chain ganglia were identical to that in the adult. Using immunocytochemistry, luteinizing hormone releasing hormone-like immunoreactivity, calcitonin gene-related peptide-like immunoreactivity, and substance P-like immunoreactivity were localized at stage III in axons within sympathetic ganglia 9 and 10. During subsequent stages, the density of fibers containing these peptides increased and it became easier to identify synaptic boutons in contact with postganglionic neurons. These observations demonstrate that projections to the lumbar sympathetic ganglia are already formed by the earliest tadpole stages, they are consistent with the previous physiological observation of nicotinic synapses in stage III ganglia, and they suggest that neuropeptide function may also begin during early stages.     

Depolarization increases vasoactive intestinal peptide- and substance P-like immunoreactivities in cultured neonatal and adult sympathetic neurons.

Depolarization has been shown to alter the biosynthesis of a number of neurotransmitters and neuromodulators. In the rat superior cervical ganglion (SCG), for example, depolarization has been reported to increase catecholamine biosynthesis and to decrease the level of substance P. We have recently found that, although the level of vasoactive intestinal peptide (VIP)-like immunoreactivity (IR) is normally low in the SCG, it increases significantly 48 hr after adult ganglia are deafferented in situ or placed in organ culture. Both manipulations decrease electrical activity of postganglionic neurons. To determine whether the increases in ganglionic VIP-IR could be a consequence of decreased depolarization of sympathetic neurons, the effect of depolarization on the expression of VIP-IR was examined in organ cultures of neonatal and adult SCG. Depolarization with elevated K+ (30 mM) or veratridine (1.5 microM) amplified, rather than blocked, the increases in VIP-IR content seen after 24 hr. Further, it increased the number of detectable VIP-IR neuronal cell bodies and processes. The stimulatory effects of veratridine were prevented by TTX. Since similar changes in expression of VIP-IR were evident in dissociated cell cultures of the SCG, cell-cell interactions requiring intact ganglionic architecture are not necessary for altered peptide expression. Elevating the concentration of Mg2+ blocked the ability of K+ and veratridine to increase VIP-IR in dissociated cell culture, raising the possibility that the effects of depolarization on VIP-IR are mediated by increased Ca2+ entry. The depolarizing conditions that increased VIP-IR also increased substance P-IR. While higher concentrations of veratridine (50 microM) blocked the elevation of both VIP- and substance P-IR induced by explantation, they produced significant neuronal death. Since depolarization with either 30 mM KCl or 1.5 microM veratridine increases expression of VIP-IR in neonatal and adult ganglia, decreased depolarization is unlikely to cause the increases in VIP- and substance P-IR that occur in culture. Furthermore, our data raise the possibility that sympathetic nerve activity in vivo can increase expression of these peptides.     

Substance P- and calcitonin gene-related peptide-like immunoreactivities in the human carotid body studied at light and electron microscopical level.

The distribution of substance P- (SP-) and calcitonin gene-related peptide-like immunoreactivities (CGRP-LI) was studied in the human carotid body at the light and electron microscopic level. Of the different compartments of the carotid body, the glomic lobules received the densest innervation by SP/CGRP double-labelled axons, followed by interlobular arteries and interlobular connective tissue. Ultrastructurally, preterminal SP/CGRP-LI axons are unmyelinated and measure 0.09-1.02 micron in diameter with a median diameter of 0.28 micron (calculated conduction velocity: 0.5 m/s). Within the glomic lobules, vesicle-containing varicosities are numerous around glomus cells, but only few of them directly contact (non-synaptically) glomus cells. The findings are consistent with the existence of two populations of SP/CGRP-LI C-fibers, one of them probably representing chemoreceptor C-fibers, the other belonging to the ubiquitous vascular sensory innervation. In addition to its localization in nerve fibers, CGRP-LI also occurs in dense core vesicles of a subpopulation of glomus cells. In this regard, the human carotid body is unique among mammalian species studied so far, which severely hampers the interpretation of pharmacological data obtained in laboratory animals in terms of human physiology.     

Effects of long-term sensory vs. sympathetic denervation on the distribution of calcitonin gene-related peptide and tyrosine hydroxylase immunoreactivities in the rat lung

In this study the effect of long-term selective sensory vs. sympathetic denervation was examined in the rat lung. Immunoreactivities for calcitonin gene-related peptide (CGRP) on the one hand and tyrosine hydroxylase (TH) on the other, were used as indexes to assess the changes in the two nerve systems. Following long-term chemosympathectomy a marked increase of CGRP-immunoreactive nerve fibers was seen in the sections, while TH-immunoreactive nerve fibers appeared depleted. Inversely, long-term sensory denervation resulted in an increase of TH-immunoreactive nerve fibers with a sharp decrease of CGRP-immunostained fibers. These results suggest a peripheral interaction between these innervation systems in the lung, the mechanism of which has still to be elucidated.     

Substance P-like immunoreactivity in human sympathetic ganglia

The substantia nigra (SN) of the cat was shown, by the anterograde and retrograde horseradish peroxidase methods, to contain neurons which send their axons to the caudomedial portions of the suprageniculate nucleus and/or lateroposterior nucleus of the thalamus; these neurons were located in the restricted region in the lateral part of the SN, which corresponds to the pars lateralis of the SN.     

Calcitonin gene-related peptide-like immunoreactive sensory fibers form synaptic contact with sympathetic neurons in the rat celiac ganglion

The present study demonstrates synaptic contact between calcitonin gene-related peptide (CGRP)-like immunoreactive axon terminals and sympathetic neurons in the rat celiac ganglion. Our observations suggest that sensory ganglion neurons directly regulate the sympathetic activity via synapses, because CGRP immunoreactive (CGRPI) fibers in this ganglion are supplied by the sensory ganglia.     

Prostacyclin enhances the evoked-release of substance P and calcitonin gene-related peptide from rat sensory neurons

Prostacyclin (PGI₂) is a potent prostanoid producing various symptoms of inflammation, including an increased sensitivity to noxious stimulation. One component of these PGI₂-mediated actions may involve activation or sensitization of sensory neurons to enhance release of neuroactive peptides. We, therefore, examined whether PGI₂ and carra prostacyclin (CPGI₂), a stable analog of PGI₂, could alter the resting and evoked release of the neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) from embryonic rat sensory neurons grown in culture. Treating isolated sensory neurons with CPGI₂ (10–1000 nM) for 30 min caused a 3-fold increase in the resting release of both peptides. One nM CPGI₂, a concentration that did not alter the resting release, significantly enhanced neuropeptide release evoked by capsaicin, 100 nM bradykinin, or 40 mM KCl. Similarly, 10 nM PGI₂ did not alter resting release, but augmented capsaicin-stimulated release of SP and CGRP 2–3 fold. In contrast, prostaglandin F_2α was ineffective in altering either resting or capsaicin-evoked peptide release. Our results demonstrate that low concentrations of PGI₂ sensitize sensory neurons to other stimuli, whereas higher concentrations evoke release directly. This PGI₂-induced augmentation of neuropeptide release may be one mechanism contributing to neurogenic inflammation.     

The actions of 3,4-diaminopyridine in bullfrog sympathetic ganglia

The effects of 3,4-diaminopyridine (3,4-DAP) on isolated sympathetic ganglia were studied by means of intracellular and extracellular recording techniques. 3,4-DAP in micromolar concentrations caused a single orthodromic stimulus to generate a brief burst of repetitive postganglionic discharges. Such stimulus-bound repetition (SBR) appeared to represent a presynaptic action of 3,4-DAP, as no repetitive firing could be evoked by antidromic or direct stimulation of ganglion cells. 3,4-DAP also increased the latency to the onset of the synaptic potential at concentrations paralleling those responsible for SBR. The actions of 3,4-DAP on synaptic transmission extended over a 10,000-fold concentration range, beginning with synaptic facilitation at micromolar concentrations (1-500 microM), and proceeding to depressant effects at millimolar concentrations (0.5-10 mM). The postganglionic repetitive discharges (SBR) induced by 3,4-DAP could be selectively suppressed by D-tubocurarine (D-Tc) or tetraethylammonium (TEA), at concentrations of these antagonists below those required to block transmission. 3,4-DAP had significant anti-curare effects, producing a four-fold shift of the D-Tc concentration-effect curve for transmission block. In contrast, 3,4-DAP neither antagonized nor enhanced the transmission block produced by TEA. These results are incompatible with traditional concepts of ganglionic blockade by D-Tc and TEA, and suggest the possibility of presynaptic sites of action in ganglion block.     

Ultrastructure and distribution of substance P-immunoreactive sensory collaterals in the guinea pig prevertebral sympathetic ganglia

A light and electron microscopic study has been made of the substance P-immunoreactive networks formed by sensory nerve fibres in the prevertebral sympathetic ganglia of the guinea pig to seek confirmation that these networks arise from collateral branches of sensory fibres passing through the ganglia and to explore the synaptic and other specialized relationships established by these networks. Slices from coeliac-superior mesenteric and inferior mesenteric ganglia of young adult males, perfusion-fixed by paraformaldehyde, were immunostained with a monoclonal antibody to substance P, and the immunolabelling was visualized by a peroxidase reaction. Immunolabelled fibres passing through the ganglia were seen by light microscopy to give off varicose collaterals that ramified in the ganglionic neuropil. Electron microscopy showed that the parent fibres were almost exclusively unmyelinated. Many collaterals ran directly beneath the basal lamina bordering the intraganglionic tissue spaces, and the varicosities either remained superficially exposed under the basal lamina or sank deeper into the supporting Schwann cells, becoming apposed to dendrites of the ganglionic neurones, upon which they formed synapses, or to other nerve terminals. The incidence of these specific associations was quantified, singly and in combination. Synapses could be situated at the same level as unlabelled synapses on the same dendrite, and exposed varicosities could lie within 0.5 micron of exposed, postsynaptic dendrites. These observations confirm a collateral, synaptic nature for the networks and suggest additional nonsynaptic modes of release and sites of transmitter action. They are consistent with the hypothesis that the system serves a nocifensor function of axon reflex type.     

Distribution of calcitonin gene-related peptide-like immunoreactivity in the nucleus ambiguus of the cat

The distribution of calcitonin gene-related peptide (CGRP) in the cat nucleus ambiguus was examined by means of a combination of horseradish peroxidase (HRP) tracing and immunohistochemical techniques. Vagal motoneurones in the nucleus ambiguus were identified by applying HRP to either the thoracic vagus or the superior laryngeal nerve or the cervical vagus. Motoneurones in the nucleus ambiguus labelled with HRP from the thoracic vagus did not contain CGRP-like immunoreactivity although CGRP-like immunoreactive cells were present in this nucleus on the same sections. In contrast, a large proportion of the motoneurones labelled from the superior laryngeal nerve and a smaller proportion of cells labelled from the cervical vagus did contain CGRP-like immunoreactivity. It is concluded that CGRP-like immunoreactivity is absent from vagal preganglionic motoneurones projecting to structures in the thorax and abdomen but is present in vagal motoneurones projecting to striated muscle of the larynx and pharynx.     

Numerous nerves with calcitonin gene-related peptide-like immunoreactivity innervate junctional epithelium of rats

We found an unusually dense concentration of nerves with calcitonin gene-related peptide-like (CGRP-L) immunoreactivity in junctional epithelium of rat gingiva. Adjacent oral epithelium was only sparsely innervated. The CGRP-L innervation may affect the local inflammatory reactions, leukocyte transmigration, and serum outflow that occur in junctional epithelium, as well as having somatosensory functions.     

Increased calcitonin gene-related peptide- and cholecystokinin-like immunoreactivities in spinal motoneurones after dorsal rhizotomy

Summary Possible changes in neuropeptides within the ventral horn of the spinal cord were investigated after unilateral dorsal rhizotomy at the lumbar level (L1–L6) in adult rats. Ten days after the surgery, immunohistochemical observations and radioimmunological determinations confirmed a marked loss of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivities within the superficial layers of the deafferented dorsal horn, as expected from the degeneration of primary afferent fibres containing these peptides. A concomitant increase in immunohistochemical staining and levels of CGRP (+296%) and CCK (+71%)- like immunoreactivities was observed in the ipsilateral ventral horn where both peptides are located in motoneurones. In contrast, substance P-like immunoreactivity that is confined to fibres and terminals within the ventral horn, was not altered by dorsal rhizotomy. These data indicate that the expression of neuropeptides in spinal motoneurones can be influenced by primary afferent inputs.     

Low calcitonin gene-related, peptide-like immunoreactivity in cerebrospinal fluid from chronic pain patients

Cerebrospinal fluid (CSF) levels of calcitonin gene-related peptide-like activity (CGRP-LI), were determined in 35 patients with painful orthopaedic disorders and the activity was compared to that of 12 healthy controls without pain. Fourteen patients had pain from osteoarthritis of the hip or the knee, 11 had rhizopathic pain due to a herniated lumbar disc and 10 had pain from a hip fracture.In all patients, decreased CGRP-LI was observed in CSF compared to the controls. The lowest values were found in the patients with osteoarthritis, while there was less, but still significant, reduction of CGRP-LI in the patients with herniated lumbar disc and those with a hip fracture. In most of the patients, CGRP-LI was also analysed at a second lumbar puncture after operative treatment, when pain had subsided or was reduced. However, the CGRP-LI remained low after treatment, which may suggest the influence of factors other than pain.     

Development of fast synaptic transmission in bullfrog sympathetic ganglia.

Extracellular recordings were made of postsynaptic responses originating in sympathetic ganglia 9 and 10 of bullfrog tadpoles and adults. At stage III, when the length and diameter of the developing hindlimb bud are equal, preganglionic stimulation elicits postganglionic action potentials in spinal nerves 9 and 10 near the sciatic plexus. Although they fluctuate in amplitude, these responses follow short trains of repetitive stimuli at 20 Hz. Their mediation by nicotinic synapses was demonstrated by reversible blockade in low Ca2+, high Mg2+ Ringer and in nicotine. Parallel sympathetic B and C systems are clearly defined by stage III. They can be selectively activated by appropriate segmental stimulation of the sympathetic chain and are characterized by distinct conduction velocities which both lie in the C fiber range (less than 1 m/s). Throughout subsequent tadpole stages, the conduction velocities of the developing B and C systems gradually double while the magnitudes of their compound action potentials grow exponentially by 100-fold. Conduction velocities reach adult values after completion of metamorphosis. These results provide physiological evidence that synapse formation in sympathetic ganglia supplying the hindlimbs begins by the earliest stages of limb bud development, is selective, and progresses over a protracted period of months, prior to myelination.     

Spinal origins of preganglionic B and C neurons that innervate paravertebral sympathetic ganglia nine and ten of the bullfrog

These experiments were designed to characterize the distribution, morphology, and number of spinal preganglionic neurons that selectively innervate the B- and C-type sympathetic neurons in paravertebral ganglia 9 and 10 of the bullfrog. For this purpose, horseradish peroxidase (HRP) was applied to the anterior end of the sectioned sympathetic chain between ganglia 8 and 9. Subsequent retrograde axonal transport of the HRP labeled ipsilateral spinal neurons whose cell bodies form a column having rostral and caudal boundaries that are, respectively, just caudal to the level of spinal nerve 4 and midway between the entry zones of spinal nerves 7 and 8. In all segments, the labeled preganglionic somata were found in the lateral half of the spinal gray and slightly dorsal to the central canal; a position analogous to that of the intermediolateral cell column in mammals. Most labeled preganglionic neurons were multipolar in shape, and the cell bodies lying between spinal nerves 4 and 5 were, on average, larger than those found between spinal nerves 7 and 8. In transverse sections that were cut near spinal nerve 5, the axons of preganglionic neurons could be seen to exit the cord through ventral roots. Counts of labeled preganglionic neurons indicate that an average +/- S.D. of 338 +/- 89 cells innervate ganglia 9 and 10. Selective labeling of preganglionic B neurons, by cutting spinal nerves 7 and 8 central to their rami communicantes at the time of HRP application, revealed an average +/- S.D. of 137 +/- 31 cells that lie exclusively between spinal nerves 4 and 6. By contrast, selective labeling of preganglionic C neurons, by cutting the sympathetic chain rostral to ganglion 7 at the time of HRP application, revealed an average +/- S.D. of 187 +/- 77 cells in an adjacent portion of the preganglionic column that is bounded by spinal nerve 6 and by a point midway between spinal nerves 7 and 8. These results thus demonstrate a clear segmental segregation between the preganglionic B and C neurons that innervate ganglia 9 and 10.     

Coexistence of calcitonin gene-related peptide and substance P-like peptide in single cells of the trigeminal ganglion of the rat: immunohistochemical analysis

The localization of calcitonin gene-related peptide (CGRP) and substance P (SP) in the rat trigeminal ganglion was examined by means of the indirect immunofluorescent method. About 40% of neurons in the ganglion contained CGRP-like immunoreactivity (CGRPI), while about 20% of neurons showed SP-like immunoreactivity (SPI). In serial sections, nearly all the SPI neurons contained CGRPI.     

Distribution of calcitonin gene-related peptide-like immunoreactivity in the brain of the lizard Podarcis hispanica

The present work studies the distribution of calcitonin gene-related peptide-immunoreactive (CGRP-li) neurons and fibers in the brain of a reptile, the lizard Podarcis hispanica. CGRP-li perikarya were not present in the telencephalon. In the thalamus, CGRP-li perikarya were restricted to the posteromedial and posterolateral nuclei. In the hypothalamus, CGRP-li cells were found mainly in the supramammillary and mammillary nuclei. In the midbrain and brainstem, CGRP-li cells appeared in the ventral tegmental area, the parabrachial nucleus, and the motor nuclei of the III-VII, IX, X, and XII cranial nerves. Motoneurons of the ventral horn of the spinal cord were also immunoreactive for CGRP. CGRP-li fibers were seen in the telencephalic hemispheres, where a dense plexus of reactive fibers appeared in the septum and in the lateral striatoamygdaloid transition area. From the latter, CGRP-li fibers entered the posterior dorsal ventricular ridge, the cell layer and deep stratum of the ventral lateral cortex, and various amygdaloid nuclei. Parts of the striatum (nucleus accumbens) and pallidum also displayed CGRP-li innervation. In the diencephalon, CGRP-li innervation was observed in parts of the dorsal thalamus and in the periventricular and medial hypothalamus. The pretectum and deep layers of the optic tectum also showed CGRP-li fibers, and numerous CGRP-li fibers were observed in the midbrain central gray, tegmentum, and pons. Some of the sensory fibers of the trigeminal, vagal, and spinal nerves were also CGRP-li. These results show that the distribution of CGRP-li structures in the reptilian brain is similar to that described for other vertebrates and suggest that the thalamotelencephalic CGRPergic projections appear to be conserved among amniote vertebrates.     

Distribution and fine structure of calcitonin gene-related peptide-like immunoreactive nerve fibers in the rat skin

Distribution of calcitonin gene-related peptide-like immunoreactive (CGRPI) nerve fibers and their fine structure were examined in the skin of rat foot pads using immunocytochemistry. The CGRPI fibers formed bundles in the dermis and subcutaneous tissue. Two types of single-stranded CGRPI fibers were seen to leave the fiber bundles: one was located along the blood vessels or around the eccrine sweat glands, while the other entered the epidermis directly or through the Meissner's corpuscles in the dermal papillae. CGRPI fibers in the epidermis were distributed widely and were occasionally associated with Merkel cells. Immunoelectron microscopic study revealed that CGRPI fibers located around blood vessels, sweat glands, epidermal keratinocytes and Merkel cells, or in the Meissner's corpuscles did not form typical synaptic contacts with underlying cells, despite being varicose and filled with vesicles resembling synaptic ones. These findings suggested that the CGRP is released non-synaptically from these terminals to influence diffusely the organs surrounding the terminals. These cutaneous fibers seemed to originate from CGRPI neurons (both small type B cells and large type A cells) in the dorsal root ganglia (DRG), because injection of fast blue dye into the cutaneous nerve resulted in labeling of these CGRPI cells in the DRG and excision of the L3-L6 DRG resulted in the non-detection of cutaneous CGRPI fibers in the foot pads. Analysis of the composition of CGRPI fibers found in the rat skin has revealed that these are mostly unmyelinated. C-type fibers with some of them being thin myelinated fibers. This was true even of CGRPI fibers at the proximal end of peripheral neurites of the DRG.(ABSTRACT TRUNCATED AT 250 WORDS)