Binding sites for calcitonin gene-related peptide in distinct areas of rat brain

Calcitonin gene-related peptide (CGRP) binding sites were localized in rat brain and spinal cord by an in vitro labeling light microscopic technique using [125I]rCGRP as radioligand. Specific rCGRP binding with a dissociation constant (Kd) of 0.53 nM to membrane preparations from rat brain cortex was characterized. The presence and the selective distribution of specific high affinity CGRP binding sites in the central nervous system suggest a role for this recently predicted peptide as a neurotransmitter.     

Calcitonin and calcitonin gene-related peptide enhance calcium-dependent potentials

Recent data suggests that calcitonin (CT) and/or calcitonin gene-related peptide (CGRP) may be potential transmitters or modulators in the nervous system. The present study analyzed the effect of CT and CGRP on the neuronal membranes of cat parasympathetic ganglia of the urinary bladder. The related peptides prolonged the duration of the afterhyperpolarization of the action potential but had no effect on resting potential or input resistance. CT and CGRP enhanced the duration of a calcium spike recorded in the presence of agents blocking Na and K channels while under similar conditions forskolin, an activator of adenylate cyclase, did not affect the calcium spike. These data suggest that the neural mechanism of action of CT and CGRP is to prolong a calcium conductance and that these effects are not mediated through cyclic AMP.     

The effect of sympathectomy on calcitonin gene-related peptide levels in the rat trigeminovascular system

The effect of sympathectomy on the calcitonin gene-related peptide (CGRP) level in the rat primary trigeminal sensory neurone was investigated. Six weeks after bilateral removal of the superior cervical ganglion there was a 70% rise in the CGRP content of the iris and the pial arteries, a 34% rise in the concentration in the trigeminal ganglion but no change in the brainstem. The CGRP rise in both end organs suggests that this phenomenon may be common to all peripheral organs receiving combined sensory and sympathetic innervations. The lack of any rise in the brainstem CGRP content raises the possibility that this process spares central terminations. In contrast, the level of neuropeptide Y, a peptide mainly contained in sympathetic terminals, fell to 35% of control values in the iris and pial arteries whilst the trigeminal ganglion and brainstem concentrations remained unchanged. The possible relevance of these observations to the clinical syndrome of postsympathectomy pain (sympathalgia) is discussed. There are similarities between the delayed onset of the human pain state and the delayed rise in sensory peptides after sympathectomy.     

Localization of calcitonin gene-related peptide in the organ of Corti of the rat: an immunohistochemical study

The localization of calcitonin gene-related peptide (CGRP)-like immunoreactive (CGRPI) structures in the cochlea was examined in the rat using immunocytochemistry. Numerous CGRPI fibers entered the organ of Corti in the intraganglionic spiral bundle and formed a dense fiber patch at the base of the inner hair cells. Much fewer, but still a significant number of CGRPI fibers were seen at the synaptic region of the outer hair cells. Since no immunoreactive cells were seen in the organ of Corti and spinal ganglion, these fibers may be one of the major components of the olivocochlear bundles originated from the superior olivary complex.     

Autoradiographic localization of calcitonin gene-related peptide binding sites in human and rat brains

125I-calcitonin gene-related peptide (CGRP) binding sites were mapped in the human brain and rat brains by in vitro macroautoradiography, and compared to each other. Binding experiments were made to characterize 125I-CGRP binding on the human and rat brains. Scatchard analysis of saturation experiments from slide-mounted sections of the human and rat cerebellum displayed 125I-CGRP binding sites with a dissociation constant (Kd) of 0.17 nM and 0.11 nM, respectively, and a maximal number of binding sites (Bmax) of 96.8 fmol/mg and 23.0 fmol/mg protein. 125I-CGRP binding was time-dependent, reversible and saturable with high affinity in the brains. Autoradiograms showed a discrete distribution of 125I-CGRP binding sites throughout the brains of human and rat with patterns similar to each other. In the human brain, the highest binding was seen in the cerebellum, inferior olivary nuclear complex, certain parts of the central gray matter, arcuate nuclei of the medulla oblongata and dorsal motor nucleus of the vagus, and densities of CGRP-binding sites were high in the nucleus accumbens, amygdala, tail of the nucleus caudatus, substantia nigra, ventral tegmental area, medial portion of the inferior colliculus, medial pontine nuclei, locus coeruleus, inferior vestibular nucleus, substantia gelatinosa of the spinal trigeminal nucleus, nucleus of the solitary tract and nucleus cuneatus lateralis. In the rat, high densities were found in the hippocampus pars anterior, nucleus accumbens, ventral and caudal portions of the nucleus caudatus-putamen, central and basolateral nuclei of the amygdala, caudal portion of the insular cortex, medial geniculate body, superior and inferior colliculi, certain portions of the central gray matter, locus coeruleus, inferior olivary nuclei, vagal complex, nucleus cuneatus lateralis and cerebellum. In contrast, in both species, most of the cortical areas including the hippocampus, most of the thalamus, and hypothalamus exhibited few binding sites. In addition, high quantities of the binding sites were seen on the pia mater and on walls of blood vessels in the brain and subarachnoidea. These results revealed essentially homologous locations of CGRP binding sites in the human and rat central nervous systems and well corresponding distributions of binding sites and endogenous CGRP-like immunoreactivity.     

Occurrence of calcitonin gene-related peptide in the chicken amacrine cells

The distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (CGRPI) in the chicken retina was investigated by means of immunohistochemistry. CGRPI was localized in the stratified amacrine cells. The terminal branching pattern of these cells was different between the central and peripheral retinal regions. Flat-mount preparations revealed these CGRPI cells to be evenly distributed in the entire retina with surprisingly rich arborization.     

Cerebral arterial innervation by nerve fibers containing calcitonin gene-related peptide (CGRP): I. Distribution and origin of CGRP perivascular innervation in the rat

The origin, density and distribution of calcitonin gene-related peptide (CGRP) immunoreactivity in cerebral perivascular nerves and the trigeminal ganglion of rats were examined in this study. CGRP immunoreactive axons were abundant on the walls of the rostral circulation of the major cerebral arteries in the circle of Willis. The fibers form a grid- or meshwork of longitudinal and circumferential axons studded with numerous varicose swellings. The density of CGRP fibers was particularly high at the bifurcation of major arteries. A few CGRP fibers cross the midline to innervate arteries on the contralateral side of the arterial tree. The arteries of the caudal circulation were sparsely innervated by CGRP fibers. In the trigeminal ganglion, about 30% of the ganglion cells had CGRP immunoreactivity. The cell size of most (75%) of CGRP neurons was less than 30 micron in diameter. There was no significant difference in staining density between small and large CGRP neurons. Unilateral transection of the maxillary and mandibular divisions of the trigeminal nerve caused a substantial decrease of CGRP immunoreactivity in the ipsilateral dorsal two-thirds of the trigeminal nucleus and cervical spinal cord but did not noticeably change the diameter of the vascular lumen or the densities of CGRP fibers in the walls of the cerebral arteries. In contrast, unilateral transection that included the ophthalmic division eliminated CGRP fibers on the ipsilateral cerebral arteries and eliminated CGRP immunoreactivity throughout the trigeminal nucleus in the brainstem and rostral cervical cord. In addition, these lesions caused a significant reduction in the diameter of the denervated arteries. The present study demonstrates that CGRP, a putative neurotransmitter/neuromodulator, is especially abundant in the rostral cerebral circulation and is derived from the ipsilateral ophthalmic division of the trigeminal nerve. In addition, the loss of CGRP perivascular nerves is associated with a reduction of the arterial lumen. This suggests that CGRP is a strong candidate as a nerve-derived trophic factor at trigeminal terminals and provides additional evidence that CGRP is a component in the trigeminovascular system influencing vascular diameter.     

Coexistence of calcitonin gene-related peptide and vasoactive intestinal peptide in cholinergic sympathetic innervation of rat sweat glands

Immunoreactivity for calcitonin gene-related peptide (CGRP) has been localized with indirect immunofluorescence techniques in the cholinergic sympathetic fibers that innervate eccrine sweat glands in the rat. This innervation also contains vasoactive intestinal peptide-like immunoreactivity (VIP-IR). A small proportion of principal neurons in stellate and lumbar sympathetic ganglia which provide innervation to the sweat glands contain detectable CGRP-immunoreactivity. The CGRP-IR neurons are immunoreactive for VIP; however, many VIP-IR neurons in these ganglia do not contain detectable levels of CGRP-IR.     

降钙素基因相关肽及内皮素A受体与蛛网膜下腔出血

Numerous studies have demonstrated that endothelin-1 combines with endothelin receptor A,resulting in intense vasoconstriction.Although calcitonin gene-related peptide(CGRP) suppresses endothelin-1,CGRP and endothelin receptor A exhibit direct biological effects on brain tissue.The present study analyzed CGRP and endothelin receptor A expression following subarachnoid hemorrhage in rabbits using immunohistochemistry.CGRP expression was significant at 5 days after model establishment,and endothelin receptor A expression was significant at 3 days after model induction.The perimeter of the basilar artery was measured to determine the amount of cerebral vasospasm.Analytical results revealed a significantly shortened basilar artery perimeter following subarachnoid hemorrhage.Changes in the basilar artery perimeter were negatively associated with endothelin receptor A expression,but positively correlated with CGRP expression in vessels.These results suggest that following subarachnoid hemorrhage,CGRP and endothelin receptor A expressions dynamically changed in brain vessels and tissues,although these changes were not synchronous.Changes in endothelin receptor A expression exhibited a significant effect on the occurrence and development of delayed cerebral vasospasm and delayed neuronal death,while CGRP relaxed vessels and protected nerves.     

Alteration of capsaicin and endotoxin-induced calcitonin gene-related peptide release from mesenteric arterial bed and spinal cord slice in 18-month-old rats

In the present study, we investigated the changes in capsaicin- and endotoxin-induced calcitonin gene-related peptide (CGRP) release from mesenteric arterial bed (MAB) and spinal cord slices (SCS) in 2-month-old and 18-month-old Wistar rats. The isolated MAB or SCS was perfused or incubated in vitro. The CGRP-like immunoreactivity in perfusate or supernatant was measured by radioimmunoassay. The results showed that endotoxin triggered CGRP release from isolated rat MAB and SCS, which represent the peripheral and central terminals of CGRP-containing sensory nerves, respectively. Either basal or stimulated CGRP release induced by capsaicin and endotoxin was significantly decreased as the rats aged from 2 to 18 months. The basal CGRP release was 14.9 ± 1.8 and 5.8 ± 1.0 pg/ml from MAB and 3.50 ± 0.54 and 1.78 ± 0.16 pg/ml/mg from SCS in 2-month-old and 18-month-old rats, respectively. The release of CGRP evoked by capsaicin (10⁻⁷ mol/L) and endotoxin (1 ≈ 5 μg/ml) from MAB and SCS was significantly decreased by more than 50% in 18-month-old rats. These data suggest that both the basal and capsaicin- or endotoxin-stimulated CGRP release from MAB and SCS display a significant decrement in aged rats that may have some physiological, pathological, and behavioural relevance in age-related diseases. J. Neurosci. Res. 53:385–392, 1998. © 1998 Wiley-Liss, Inc.     

Immunohistochemical evidence for the coexistence of calcitonin gene-related peptide- and choline acetyltransferase-like immunoreactivity in neurons of the rat hypoglossal, facial and ambiguus nuclei

The present immunocytochemical study demonstrates that calcitonin gene-related peptide-like immunoreactivity (CGRPI) coexists with acetylcholine in single cells of hypoglossal, facial and ambiguus nuclei. The experiments were done using alternate frozen sections from relevant regions of the rat brain. We further show that CGRPI is localized in the nerve terminals that form neuromuscular junctions in the tongue muscles.     

Denervation-induced intraspinal synaptogenesis of calcitonin gene-related peptide containing primary afferent terminals

The purpose of the present study is to provide evidence that chronic spinal denervation leads to an increase in numbers of synaptic terminals from a specific population of primary afferent fibers. Rats were unilaterally deafferented for 35 days (chronic denervation) by dorsal rhizotomies performed from T2 to T8 and T10 to L5, which isolates or spares the T9 root. The contralateral T9 root was spared by similar surgery 5 days (acute denervation) prior to sacrifice. The survival time on the chronic side presumably allows sprouting of T9 primary afferents to occur, whereas the time on the acute side does not. The terminals were labeled with calcitonin gene-related peptide (CGRP), which is a compound that labels a specific population of primary afferent fibers and terminals, and stereological methods were used to determine the numbers of immunolabeled terminals in laminae I and IIo on the chronic and acute sides of the T9 spinal cord. The findings are that the chronic side had approximately twice as many terminals as the acute side. This difference is statistically significant. These findings are compatible with the hypothesis that chronic denervation leads to synaptogenesis from surviving primary afferent fibers.     

降钙素基因相关肽对大鼠局灶性脑缺血保护作用的研究

线栓法建立大鼠局灶脑缺血动物模型，定量研究降钙素基因相关肽（CGRP）对大鼠局灶脑缺血体积的影响。结果：模型建立前1小时使用CGRP预防性治疗可明显减小脑缺血体积，与对照组比较减幅达54％（P＜0．01）；模型建立后2小时CGRP治疗组脑缺血体积仅比对照组减少12％（P＞0．05）。提示：CGRP对缺血神经组织有保护作用，但治疗时间窗较短。     

降钙素基因相关肽与内皮素受体A在兔实验性蛛网膜下腔出血后脑组织表达的对照研究

目的探讨降钙素基因相关肽(CGRP)和内皮素受体A(ETRA)在蛛网膜下腔出血(SAH)后脑组织中的表达情况及意义。方法日本大耳白家兔45只,随机分为SAH模型组(20只),盐水对照组(15只),穿刺对照组(5只),空白对照组5只。采用枕大池2次注血法制作SAH的动物模型,并进行CGRP和ETRA的免疫组化染色,观察CGRP和ETRA的免疫表达。结果CGRP和ETRA免疫阳性标记在SAH组模型均有表达,CGRP阳性细胞在1h时少量表达,3～7d时表达明显,其中5d时最显著,10d时阳性细胞表达已经减少,但未达到正常水平。SAH模型组ETRA主要表达在神经元和胶质细胞胞浆内,建模后1h时染色较淡;3d时染色最强烈;5d时染色较3d弱,此后染色逐渐变淡。结论SAH后脑组织CGRP和ETRA的表达增强,并呈现明显的动态改变,但两者的变化趋势并不同步;CGRP和ETRA可能在延迟性神经功能障碍的发生与发展中起重要作用。     

Local anesthetics inhibit veratridine-induced secretion of calcitonin gene-related peptide (CGRP) from cultured rat trigeminal ganglion cells

Secretion of calcitonin gene-related peptide (CGRP) was studied with the model system of dispersed adult rat trigeminal ganglion cells. Veratridine stimulated secretion of CGRP immunoreactivity. Tetrodotoxin and local anesthetics inhibited veratridine-stimulated peptide secretion. These observations implicate sodium channels in CGRP secretion and are consistent with a role for the peptide as an extracellular neuromodulator in the sensory nervous system.     

Rat facial motoneurons express increased levels of calcitonin gene-related peptide mRNA in response to axotomy

The expression and localization of the mRNA encoding the calcitonin gene-related peptide (CGRP) were analyzed in the rat facial nucleus after axotomy by Northern blot analysis and by in situ hybridization histochemistry (ISH) using a synthetic 32P-labeled oligonucleotide probe. Northern blot analysis revealed the presence of the 1.2 kb CGRP mRNA in RNA extracted from the facial nucleus. This mRNA species was strongly increased after axotomy of the facial nerve. By ISH increased levels of CGRP mRNA were observed as soon as 16 hr after axotomy compared with the unoperated nucleus. CGRP mRNA could be localized in more than 50% of the motoneurons. Three populations of motoneurons with no, moderate, or strong labeling for CGRP mRNA could be distinguished. Peak expression of CGRP mRNA during the first 48 hr was followed by a decline to moderate levels at day 4 after lesion, and to almost basal levels at days 7 and 9. These data demonstrate that axotomy of the facial nerve leads to an early and strong induction of CGRP gene expression in motoneurons of the facial nucleus.     

Light and electron microscopic studies of calcitonin gene-related peptide-like immunoreactive neurons and axon terminals of the nucleus of the tractus solitarius of the rat

This study was an examination of the ultrastructural characteristic features of calcitonin gene-related peptide (CGRP)-like immunoreactive neurons and their axon terminals in the nucleus of the tractus solitarius of the rat. Some axon terminals were identified as receiving synaptic inputs from non-immunoreactive axon terminals. This may suggest that part, if not all, CGRP containing afferents are affected presynaptically by other afferents.     

The localization and release of substance P and calcitonin gene-related peptide at nerve fibre endings in rat cutaneous nerve neuroma

This study uses radiological and immunocytochemical techniques to investigate the localization, content, transport and release of substance P- and calcitonin gene-related peptide-like immunoreactivity (SP-LI and CGRP-LI, respectively) in nerve fibre endings in 1-, 3- and 5-week-old cutaneous nerve neuromas. Neuromas were induced by ligating and transecting the saphenous nerve in anaesthetized Sprague-Dawley rats. The content of both neuropeptides in 3-week-old saphenous nerve neuroma was significantly reduced compared to that in normal saphenous nerve. At 5 weeks the levels of the peptides in the neuromas had returned to normal but remained reduced in the nerve just proximal to the neuroma. Following a 24-h ligation of the nerve proximal to 3-week-old neuromas there was a diminished immunocytochemical staining for SP-LI and CGRP-LI both proximal and distal to the ligature when compared to that seen at ligations of normal nerves. This indicates a decreased transport of the neuropeptides both to and from the 3-week-old neuromas. The density of neuropeptide staining at ligatures of nerves with 5 week or older neuromas had increased, but still remained less than that seen at ligations of normal nerve. Both a basal and a bradykinin-induced release of SP-LI and CGRP-LI from nerve fibre endings in the neuroma was demonstrated. The basal release was demonstrated by exposing the neuromas, in situ, to solutions containing 50 μM morphine plus 2 mM CoCl₂ for 24 h. In 5-week-, but not 3-week-old neuroma this produced an increase in the content of SP-LI, and resulted in the appearance of SP-LI and CGRP-LI-containing fibres with varicosities in the neuromas. A stimulated release of both neuropeptides from nerve fibre endings in 5-week-old saphenous nerve neuromas could be demonstrated by superfusing the neuromas with 90 μM bradykinin. This produced a significant increase of SP-LI and CGRP-LI in the superfusate. The amount of SP-LI released from 3-week neuromas was less than that released from 1- and 5-week-old neuromas. These changes in neuropeptide content and release are discussed in relation to the regenerative capabilities of damaged nerve fibre endings.     

Transient receptor potential channel A1 involved in calcitonin gene-related peptide release in neurons

Transient receptor potential channel A1 is one of the important transducers of noxious stimuli in the primary afferents, which may contribute to generation of neurogenic inflammation and hyperalgesia. The present study was designed to investigate if activation of transient receptor potential channel A1 may induce calcitonin gene-related peptide release from the primary afferent neurons. We found that application of allyl isothiocyanate, a transient receptor potential channel A1 activator, caused calcitonin gene-related peptide release from the cultured rat dorsal root ganglion neurons. Knock- down of transient receptor potential channel A1 with an antisense oligodeoxynucleotide prevented calcitonin gene-related peptide release by allyl isothiocyanate application in cultured dorsal root ganglion neurons. Thus, we concluded that transient receptor potential channel A1 activation caused calcitonin gene-related peptide release in sensory neurons.