Axotomy alters putative neurotransmitters in visceral sensory neurons of the nodose and petrosal ganglia.

Acute peripheral axotomy of the visceral sensory neurons of the vagus and glossopharyngeal nerves removes peripheral depolarizing and trophic influences to their sensory ganglia. To study axotomy-induced changes in the putative neurotransmitters of visceral sensory neurons, rats were sacrificed 1, 3, 7 or 14 days after transection of either the cervical vagus and superior laryngeal nerves (to affect peripheral axotomy of the nodose ganglion) or the glossopharyngeal and carotid sinus nerves (to affect peripheral axotomy of the petrosal ganglion). The numbers of tyrosine hydroxylase (TH)-immunoreactive (ir), vasoactive intestinal peptide (VIP)-ir, calcitonin-gene-related peptide (CGRP)-ir, and substance P (SP)-ir neurons in the respective ganglia were analyzed in axotomized and control ganglia. In the nodose ganglion, axotomy of the cervical vagus resulted in a rapid (by 1 day) reduction in the number of TH-ir cells, whereas VIP-ir neurons were dramatically increased in number by 3 days. CGRP- and SP-ir cells in the nodose ganglion were relatively unaffected by axotomy. In the petrosal ganglion, axotomy of the glossopharyngeal and carotid sinus nerves greatly reduced the number of TH-ir cells but did not alter the number VIP-ir neurons. CGRP- and SP-ir neurons in the petrosal ganglion were reduced in number by axotomy. Thus, axotomy of visceral sensory neurons differentially changed the content and perhaps the expression of putative transmitters. Differential changes were seen among transmitters in a single ganglia and between ganglia. These data demonstrate the plasticity of putative neurotransmitter systems in visceral afferent systems of adult rats.     

Calcitonin gene-related peptide- and substance P-containing primary afferent fibers in the dorsal column of the rat

The dorsal column and its nuclei exhibit a considerable number of fibers containing neuropeptides, such as calcitonin gene-related peptide (CGRP) and substance P (SP), whose origins and functional roles are as yet unknown. The present study attempts to determine the origin and nature of these fibers by means of immunohistochemistry combined with several experimental manipulations. A similar study was done on scattered substance P (SP) fibers whose presence was confirmed in this study. Transection of the upper cervical cord of rats resulted in an accumulation of CGRP, sometimes with SP also, in the caudal aspect of the lesion, thus indicating the presence of peptide-containing ascending fibers. Hemitransection of the dorsal column at the level of C2-3 caused reduction of CGRP-containing fibers in the dorsal column and its nuclei on the operated side. Electron microscopic observation of the nucleus gracilis revealed that CGRP-like immunoreactive terminals made direct axodendritic synaptic contacts. Medium- to large-sized neurons in the dorsal root ganglia were labeled with Fast blue dye which was injected into the dorsal column nuclei. These included medium- to large-sized neurons exhibiting immunoreactivity to CGRP-like substances, and neurons of a medium size which were immunoreactive to SP-like compounds. The incidence of the former was higher at the thoracic level than at the cervical and lumbar levels, while that of the latter was very low. Electron microscopic observation of CGRP-containing fibers in the cervical region of the dorsal column revealed that 88% of these fibers were unmyelinated and the remainder were thinly myelinated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coexistence of S100β and putative transmitter agents in vagal and glossopharyngeal sensory neurons of the rat

The coexistence of S100β with calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM), nicotinamide adenosine dinucleotide phosphate-diaphorase (NADPH-d), and tyrosine hydroxylase (TH) was examined in the glossopharyngeal and vagal sensory ganglia. S100β immunoreactive (-ir) neurons in the jugular and petrosal ganglia frequently colocalized CGRP- or SP-ir, whereas S100β-ir neurons in the nodose ganglion infrequently contained CGRP- or SP-ir. No S100β-ir neurons in the jugular and petrosal ganglia showed SOM-ir while the small number of SOM-ir neurons in the nodose ganglion colocalized S100β-ir. Many neurons in the nodose ganglion colocalized S100β-ir and NADPH-d activity, whereas S100β-ir neurons in the jugular and nodose ganglia infrequently contained NADPH-d activity. S100β- and TH-ir were frequently colocalized in nodose ganglion but not in petrosal or jugular ganglion neurons. These findings suggest relationships between S100β and specific putative transmitters in functions of subpopulations of vagal and glossopharyngeal sensory neurons.     

Adrenalectomy increases the number of substance P and somatostatin immunoreactive nerve cells in the rat lumbar dorsal root ganglia

Using an immunocytochemical technique we have analyzed changes in substance P, somatostatin, calcitonin gene-related peptide, and galanin immunoreactivity pattern in the rat dorsal root ganglia. After 7 days of adrenalectomy, sham operated rats were compared with adrenalectomized animals either receiving a daily intraperitoneal injection of 10 mg/kg b.wt. corticosteronev or vehicle. Three lumbar ganglia from each animal were blocked, serially cut, and immunostained for each neuropeptide by means of the biotin-avidin-peroxidase technique. A systematic sampling of immunoreactive ganglion cells was performed and the sample number of immunoreactive ganglion cells was calculated. After adrenalectomy, the number of substance P and somatostatin immunoreactive ganglion cells markedly increased ((means±S.E.M.): 245 ± 68versus123 ± 12 for sham operated animals, P < 0.01 (substance P) and 42 ± 8 as compared to 22 ± 9 for sham operated animals, P < 0.01 (somatostatin)). No significant changes were found in the number of calcitonin gene-related peptide and galanin immunoreactive cells after adrenalectomu. These results suggest that adrenal steroid hormones may reduce the synthesis of both substance P and somatostatin in the dorsal root ganglion cells. Daily treatment with a high dose of corticosterone, mimicking its serum levels after stress, failed to prevent the increase of peptide contents after adrenalectomy. These observations also indicate that a tonic action of corticosterone on mineralocorticoid receptors may be crucial for peptide regulation in the spinal ganglia. These results may be of relevance to adrenalectomy induced changes in sensory mechanisms, neurogenic inflammation and pain transmission and to a role of substance P and somatostatin in these processes.     

The effect of streptozotocin-induced diabetes mellitus on substance P and calcitonin gene-related peptide expression in the rat trigeminal ganglion

Substance P (SP) and calcitonin gene-related peptide (CGRP) constitute the main sensory peptides in the trigeminal ganglion (TG). The objective of this study was to characterize peptidergic changes in the streptozotocin-induced diabetes mellitus rat model both quantitatively and qualitatively. Diabetes mellitus was induced by a single intraperitoneal injection of streptozotocin (65 mg/kg) and the levels of SP and CGRP were measured by means of radioimmunoassay (RIA) in a time-dependent manner. Peptide immunoreactivities were characterized by high pressure liquid chromatography (HPLC). The expression of both neuropeptides was examined 5 weeks after streptozotocin injection using in situ hybridization with 35S-labelled oligonucleotides. Saline-injected rats served as controls. SP was significantly decreased in the diabetic rat TG, i.e. , a 44.6% (+/-10.9) decrease after 1 week, 40.2% (+/-11.8) after 3 weeks and 72.3% (+/-14.6) after 5 weeks. CGRP was decreased only after 5 weeks (19.6% decrease +/-3.9), whereas at later stages, both peptide levels returned to normal values. HPLC revealed one major peak coeluting with the synthetic peptides. By using in situ hybridization, a significantly increased signal of both peptide-encoding mRNAs was found (43.8%), which seems to act to restore a diabetes-associated depletion of neuropeptides in the diabetic rat TG. The decreased SP- and CGRP levels in the diabetic rat TG reflect a diabetes-associated deficit which may be clinically relevant. Diabetes mellitus is associated with a variety of ocular complications, even corneal complications, including decreased corneal sensitivity, which in many ways resemble those after interruption of the normal trophic innervation of the eye. Our results point to reduced availability of neuropeptides for corneal innervation and may thus support the idea of a partial loss of trophic influences from the trigeminal nerve in diabetics.     

Peptide coexistence in axon terminals within the superficial dorsal horn of the rat spinal cord.

The somata of primary sensory neurons have been shown to contain up to four (and possibly more) neuroactive peptides. Although each of these peptides has been separately located in axon terminals within the superficial dorsal horn of the spinal cord, it is not clear whether multiple peptide coexistence is also a feature of terminal varicosities. The aim of this study was to determine whether the peptides substance P (SP) and calcitonin gene-related peptide (CGRP), which are colocalized in the somata of a large number of primary sensory neurons, coexist in the central terminals of these neurons in the spinal cord. The protein A-gold technique of antigen localization was used to screen single boutons in laminae I and II of the rats spinal cord for SP- and CGRP-like immunoreactivity at the ultrastructural level. The results show that SP and CGRP are colocalized within a large number of synaptic boutons in the superficial dorsal horn. Furthermore, evidence was obtained to suggest that both SP and CGRP may be found in the same synaptic vesicle within these boutons. These findings indicate that both SP and CGRP may be coreleased from single terminals in the superficial dorsal horn. This is of considerable interest in view of the reported interaction between SP and CGRP in nociceptive behavioral responses in the rat.     

Modulatory effects of calcitonin gene-related peptide and substance P on human cholinergic sweat secretion

Immunoreacttvtty to various peptides has been demonstrated in nerve terminals around the sweat glands, suggesting a regulatory function for these peptides on sweating. The present study evaluated the calcitonin-gene related peptide and substance P related regulation of sweating in man. Both calcitonin-gene related peptide and substance P, when administered alone, failed to cause sweat secretion, whereas sweating induced by methacholine chloride alone was four times greater when administered with calcitonin-gene related peptide and suppressed by 70% when administered with substance P. The degree of calcitonin-gene related peptide dependent augmentation and substance P dependent suppression of the methacholine chloride induced sweating was dependent on the concentration of calcitonin-gene related peptide and substance P. These findings suggest that calcitonin-gene related peptide enhances cholinergic sweating and substance P inhibits it.     

Distribution of substance P and calcitonin gene-related peptide immunoreactive nerve fibers in the trachea of chronically hypoxic rats

The distribution of substance P and calcitonin gene-related peptide immunoreactive nerve fibers in the trachea was compared between normoxic and chronically hypoxic rats (at 380 mm Hg for 10 weeks). In the normoxic trachea, the immunoreactivity to either peptide was seen in the nerve fibers in four principal locations: a) within and b) under the ciliated epithelium, c) within the smooth muscle bundles in the posterior wall, and d) in the connective tissue and around blood vessels in the lamina propria and submucosa. These immunoreactive fibers within the epithelium and smooth muscle bundles, in the connective tissue, and around blood vessels were thin and displayed some varicosities, and those under the epithelium appeared as thick nerve bundles. When the distribution and density of immunoreactive fibers were compared between normoxic and chronically hypoxic tracheas, there was a difference in number of substance P and calcitonin gene-related peptide immunoreactive fibers penetrating into the epithelium, although there was no difference in the other three locations. The mean number of substance P and calcitonin gene-related peptide immunoreactive intraepithelial fibers per section of the chronically hypoxic trachea was significantly increased. Because substance P and calcitonin gene-related peptide are predominant signal peptides of primary sensory neurons, the increase of substance P and calcitonin gene-related peptide immunoreactive fibers suggests that altered airway reflexes may be a feature of hypoxic adaptation.     

Ontogeny of sensory and autonomic nerves in the developing mouse skeleton

Background

Bone innervation is implicated in bone modeling and remodeling. This study investigates skeletal nerve development in embryonic and newborn mice, focusing on sensory and autonomic nerves and their temporal occurrence.

Materials and methods

The ontogeny of innervation and angiogenesis in the hindlimb skeleton of mice was studied from embryonic day (E) 15 to postnatal day (P) 20. Neuronal tissue was immunohistochemically labeled for detection of growth associated protein 43 (GAP-43), protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), and neuropeptide Y (NPY). Vascular endothelium was labeled for platelet endothelium cell adhesion molecule-1 (PECAM-1). Morphology was evaluated with hematoxylin and eosin staining.

Results

GAP-43, PGP 9.5, CGRP, and PECAM-1 were all present at E 15, adjacent to areas with high osteogenic and chondrogenic activity. In the primary ossification centers, GAP-43 was found at E 15, PGP 9.5 at E 17, CGRP at E 19, and NPY at P 4. The same time lag in appearance was observed in the secondary ossification centers. The covering capillary network was initially dense, but became mature and sparse from P 12 onwards.

Conclusion

A functional nerve supply co-localized with a rich capillary network is seen early in the developing mouse skeleton, especially in areas with high osteogenic activity. Sensory innervation occurs prior to partus, while autonomic innervation (revealed by the presence of NPY and TH) is established post partum. The findings indicate a time-related development of nerves with different qualities, according to skeletal development.     

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.     

Significant changes in neuropeptide concentrations in the brain of normotensive (WKY) and spontaneously hypertensive (SHR) rats following knee joint monoarthritis

Changes induced by chronic monoarthritis in the nervous system was studied by measuring concentrations of substance P (SP)-, neurokinin A (NKA)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-like immunoreactivities in the brain and in the knee joints of control and monoarthritic normotensive (WKY) and spontaneously hypertensive (SHR) rats on day 21 after the induction of monoarthritis. Knee joint monoarthritis was induced by intra-articular injection of Freund's adjuvant into the right knee joint. The severity of arthritis was examined by measuring knee volumes and scratching behaviour and by X-ray. The right knee of both WKY and SHR monoarthritic rats had an increased volume and osteoporosis. SHR rats had more severe arthritis and increased scratching behaviour compared to the WKY. Tachykinins were significantly decreased in the hypothalamus of arthritic rats. In the pituitary higher concentrations of tachykinins and CGRP were found in the arthritic and/or control SHR rats than in the WKY. In the occipital cortex, striatum and hippocampus NPY was increased in monoarthritic rats. No correlation was found between neuropeptide concentrations in the brain and knee joints. Decrease of tachykinins and increase of CGRP to different degree in the hypothalamus and/or pituitary of the arthritic WKY and SHR rats indicates that these changes were selectively associated with the basal level of sympathetic tone and possibly related to the greater severity seen in SHR rats. The increase of NPY in the brain, not influenced by sympathetic tone, may be part of a general defence reaction to inflammation.     

The coexistence of TrkA with putative transmitter agents and calcium-binding proteins in the vagal and glossopharyngeal sensory neurons of the adult rat

The presence of the neurotrophin receptor, TrkA, in neurochemically identified vagal and glossopharyngeal sensory neurons of the adult rat was examined. TrkA was colocalized with calcitonin gene-related peptide (CGRP), parvalbumin, or calbindin D-28k in neurons of the nodose, petrosal and/or jugular ganglia. In contrast, no TrkA-immunoreactive (ir) neurons in these ganglia colocalized tyrosine hydroxylase-ir. About one-half of the TrkA-ir neurons in the jugular and petrosal ganglia contained CGRP-ir, whereas only a few of the numerous TrkA-ir neurons in the nodose ganglion contained CGRP-ir. Although 43% of the TrkA-ir neurons in the nodose ganglion contained calbindin D-28k-ir, few or no TrkA-ir neurons in the petrosal or jugular ganglia were also labeled for either calcium-binding protein. These data show distinct colocalizations of TrkA with specific neurochemicals in vagal and glossopharyngeal sensory neurons, and suggest that nerve growth factor (NGF), the neurotrophin ligand for TrkA, plays a role in functions of specific neurochemically defined subpopulations of mature vagal and glossopharyngeal sensory neurons.     

Regenerative effect of human recombinant NGF on capsaicin-lesioned sensory neurons in the adult rat

Nerve growth factor (NGF) has the ability to increase the content of peptide transmitter in intact primary sensory afferents of the adult rat. We have previously shown that NGF can also induce a refill of peptide transmitters in capsaicin-depleted peptidergic nerve terminals of the rat paw skin upon intraplantar injection. The present study was aimed at investigating the neurochemical, immunohistochemical and functional recovery of peripheral and central terminals of capsaicin-lesioned afferents following administration of recombinant human NGF-β (rhNGF-β). The systemic capsaicin treatment in adult rats by 50 mg/kg s.c. (day 0) was followed by intraplantar rhNGF-β injections (4 μg each) into one hind paw on days 1, 2, 3, 5, 6 and by the analysis on day 8. The content of the marker peptide calcitonin gene-related peptide (CGRP) showed a 100% NGF-induced recovery in the peripheral (sciatic nerve) and central axons (lumbar dorsal roots) on the side of the NGF treatment and also in the contralateral sciatic nerve and lumbar dorsal roots. In the terminals of the hind paw skin, the recovery of the CGRP content, as measured by radioimmunoassay, was 100% in the plantar and 80% in the dorsal skin ipsilaterally, and 55% in the dorsal and plantar hind paw skin contralaterally. In the lumbar dorsal spinal cord, CGRP content recovered by 85% bilaterally. The morphological appearance of the sensory nerve terminals was visualized by CGRP-immunohistochemistry. In the paw skin, the CGRP-immunoreactive (CGRP-IR) nerve endings were restricted to a fragmentary subepidermal plexus after the capsaicin treatment, whereas the subsequent NGF treatment caused a bilateral recovery of the subepidermal plexus and an intact reinnervation of the epidermis and blood vessels with free nerve terminals. The capsaicin-induced fragmentation of the CGRP terminal plexus in laminae I and II of the lumbar spinal dorsal horn was also markedly repaired on both sides by the intraplantar NGF injections. The NGF treatment caused the CGRP nerve terminals in the spinal cord to regain their ability of releasing transmitter upon capsaicin stimulation as shown in tissue slice superfusion experiments. These results show that within one week, rhNGF-β can induce a complete reinnervation of skin and spinal cord with intact CGRP-IR nerve terminals after an acute capsaicin lesion.     

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.     

Origins and distribution of calcitonin gene-related peptide-containing nerves in the wall of the cerebral arteries of the guinea pig with special reference to the coexistence with substance P

The origins and overall distribution of calcitonin gene-related peptide-like immunoreactivity (CGRPI) in the wall of the cerebral arteries were investigated in the guinea pig by using whole-mounts. Two types of CGRPI fibers were seen; one forming dense fiber bands, located among the periadventitial nerves, and the other forming a meshwork. CGRPI fibers in the periadventitial nerves often leave these nerves to form a meshwork, of a density that varies according to the diameter or location of the blood vessel. The present study showed that CGRPI fibers in the walls of the carotid arterial system originated from the trigeminal ganglion, and those in the vertebrobasilar arterial system from other origins besides the trigeminal ganglion. We also examined the coexistence of this peptide with substance P-like immunoreactive (SPI) structures in a single neuron system. Double staining immunocytochemistry showed that the patterns of the running of CGRPI and SPI fibers in the wall of the cerebral arteries were similar, and this method also demonstrated the presence of neurons containing both CGRPI and SPI structures in single cells of the trigeminal ganglion, which is the major origin of these fibers in the cerebral arteries.     

Coexistence of calbindin D-28k and NADPH-diaphorase in vagal and glossopharyngeal sensory neurons of the rat

The presence and coexistence of calbindin D-28k-immunoreactivity (ir) and nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase activity (a marker of neurons that are presumed to convert L-arginine to L-citrulline and nitric oxide) were examined in the glossopharyngeal and vagal sensory ganglia (jugular, petrosal and nodose ganglia) of the rat. Calbindin D-28k-ir nerve cells were found in moderate and large numbers in the petrosal and nodose ganglia, respectively. Some calbindin D-28k-ir nerve cells were also observed in the jugular ganglion. NADPH-diaphorase positive nerve cells were localized to the jugular and nodose ganglia and were rare in the petrosal ganglion. A considerable portion (33–51%) of the NADPH-diaphorase positive neurons in these ganglia colocalized calbindin D-28k-ir. The presence and colocalization of calbindin D-28k-ir and NADPH-diaphorase activity in neurotransmitter-identified subpopulations of visceral sensory neurons were also studied. In all three ganglia, calcitonin gene-related peptide (CGRP)-ir was present in many NADPH-diaphorase positive neurons, a subset of which also contained calbindin D-28k-ir. In the nodose ganglion, many (42%) of tyrosine hydroxylase (TH)-ir neurons also contained NADPH diaphorase activity but did not contain calbindin D-28k-ir. These data are consistent with a potential co-operative role for calbindin D-28k and NADPH-diaphorase in the functions of a subpopulation of vagal and glossopharyngeal sensory neurons.     

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.     

Regional distribution of Neuropeptide K and other tachykinins (Neurokinin A, Neurokinin B and substance P) in rat central nervous system

Neurokinin A and B (NKA and NKB) and neuropeptide K (NPK) were recently isolated from porcine spinal cord and brain, and together with substance P (SP) considerably extend the list of tachykinin-like peptides present in the mammalian nervous system. In order to investigate the distribution of tachykinins in the central nervous system (CNS) we have recently developed sensitive radioimmunoassays (RIA) for both SP and NKA. As NKB and NPK cross-react in the RIA for NKA we were able to determine the content of NKA, NKB and NPK after separation of rat CNS extracts by reverse-phase high-performance liquid chromatography (HPLC). Comparison of different extraction methods suggested that 0.1 M hydrochloric acid gave the best extraction and recovery of NKA-like immunoreactivity from rat brain. Characterization of these tachykinins using HPLC and gel-filtration columns revealed that C18 columns did not adequately separate NPK from NKB under the conditions used by previous authors. Thus 'NKB' content reported previously on the basis of HPLC separation would correspond to the sum of both NPK and NKB content. In the present study, therefore, we introduced modified elution conditions to resolve NPK from NKB and determined the regional distributions of these tachykinins in the rat CNS. SP was the most abundant tachykinin in every region studied. After SP, the NKA concentration was highest and NKB concentration was lowest in all regions except for the cortex and hippocampus where the NPK concentration was highest. The molar ratio of these peptides seemed to be relatively constant in the 3 regional groups (striatum-substantia nigra, cerebral cortex-hippocampus, dorsal root ganglia-dorsal and ventral horns of spinal cord) and suggests that regional specific translation or processing may exist.     

Effects of substance P and calcitonin gene-related peptide on axonal transport in isolated and cultured adult mouse dorsal root ganglion neurons

Substance P and calcitonin gene-related peptide (CGRP) released from primary sensory neurons are known to play important roles in nociception and nociceptive transmission. In the present study, we attempted to clarify the roles of these neuropeptides in the regulation of axonal transport in sensory neurons. Cells were isolated from adult mouse dorsal root ganglia and cultured in F-12 medium containing fetal bovine serum for 48 h until their neurites were grown. These isolated and cultured DRG cells were mostly (>98%) small (diameter <25 microm) and medium (diameter, 25-40 microm) in size, and were immunoreactive for substance P and CGRP (85.9 and 66. 0% of total cells, respectively). Video-enhanced microscopy was applied to observe particles transported within neurites. Application of substance P (100 nM) decreased the number of particles transported in both anterograde and retrograde directions in each of DRG neurons tested (n=5). The instantaneous velocities of individual particles transported in anterograde and retrograde directions were also reduced by substance P. In contrast, alpha-CGRP (100 nM) increased the number of particles transported in both directions in each of DRG neurons tested (n=5), and also increased the instantaneous velocities of particles transported bidirectionally. Application of beta-CGRP (100-1000 nM) did not elicit any effect on axonal transport. Therefore, axonal transport in sensory neurons seems to be modulated by substance P and alpha-CGRP, both of which can be derived from its own and adjacent sensory neurons.     

Calcitonin gene-related peptide promotes mechanical nociception by potentiating release of substance P from the spinal dorsal horn in rats

In vitro superfusion with capsaicin (5 X 10(-7) M) of slices of the dorsal half of the rat spinal cord produced a significant increase in a release of immunoreactive substance P (iSP). Calcitonin gene-related peptide (CGRP: 10(-6) M) significantly potentiated the capsaicin-induced release of iSP. On the other hand, when CGRP (5 nmol/rat) was intrathecally injected, the peptide produced a significant hyperalgesia to mechanical noxious stimuli (pinching the hind paw), but aversive responses and potentiation of substance P-induced aversive responses were never observed. These findings suggest that in the rat spinal dorsal horn, CGRP potentiates the release of substance P from the primary afferent terminal and promotes the transmission of nociceptive information induced by mechanical noxious stimuli.