Neuronal pathways to the rat thyroid revealed by retrograde tracing and immunocytochemistry

The distribution and origin of the nerve fibres innervating the rat thyroid were studied by immunocytochemistry, retrograde tracing and denervation experiments. Immunocytochemistry revealed nerve fibres containing noradrenaline, neuropeptide Y, vasoactive intestinal peptide, peptide histidine-isoleucine, galanin, substance P, neurokinin A and calcitonin gene-related peptide around blood vessels and follicles. Many of these transmitter candidates were found to co-exist with each other in different combinations in different subpopulations of neurons. Sympathectomy eliminated all noradrenaline- and noradrenaline/neuropeptide Y-containing fibres in the thyroid. Cervical vagotomy eliminated about 50% of the galanin-, substance P- and calcitonin gene-related peptide-containing fibres. Local denervation (removal of the thyroid ganglion and the thyroid nerve) eliminated all galanin- and substance P-immunoreactive fibres and the majority of noradrenaline-, noradrenaline/neuropeptide Y-, vasoactive intestinal peptide- and calcitonin gene-related peptide-containing fibres in the thyroid gland. Injection of True Blue into the thyroid gland labelled cell bodies in the thyroid ganglion, the laryngeal ganglion, the superior cervical ganglion, the jugular-nodose ganglionic complex, the dorsal root ganglia (C2-C5) and the trigeminal ganglion. Judging from the number of labelled nerve cell bodies, the superior cervical ganglion and the thyroid ganglion contribute most to the thyroid innervation, while the laryngeal ganglion and the trigeminal ganglion contribute least. The True Blue-labelled ganglia were examined for the presence of various populations of nerve cell bodies (only major populations are listed). The thyroid ganglion harboured neuropeptide Y, vasoactive intestinal peptide and galanin/vasoactive intestinal peptide cell bodies (in order of predominance); the laryngeal ganglion galanin/vasoactive intestinal peptide, vasoactive intestinal peptide and calcitonin gene-related peptide cell bodies; the superior cervical ganglion noradrenaline/neuropeptide Y and noradrenaline cell bodies; the jugular ganglion calcitonin gene-related peptide, substance P/calcitonin gene-related peptide and galanin/substance P/calcitonin gene-related peptide cell bodies; the nodose ganglion vasoactive intestinal peptide and vasoactive intestinal peptide/galanin cell bodies; the dorsal root ganglia (C2-C5) and the trigeminal ganglion calcitonin gene-related peptide, substance P/calcitonin gene-related peptide and galanin/substance P/calcitonin gene-related peptide cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of peripheral nerve injury on dorsal root ganglion neurons in the C57 BL/6J mouse: marked changes both in cell numbers and neuropeptide expression

Several types of changes have been reported to occur in dorsal root ganglia following peripheral nerve injury, including loss of neurons and increases and decreases in peptide expression. However, with regard to loss of neurons, results have not been consistent, presumably due to different quantitative methodologies employed and species analyzed. So far, most studies have been conducted on rats; however, with the fast development of the transgenic techniques, the mouse has become a standard model animal in primary sensory research. Therefore we used stereological methods to determine the number of neurons, as well as the expression of galanin message-associated peptide, a marker for galanin-expressing neurons, neuropeptide Y, and calcitonin gene-related peptide in lumbar 5 dorsal root ganglia of both control C57 BL/6J mice and in mice subjected to a 'mid-thigh' sciatic nerve transection (axotomy). In control animals the total number of lumbar 5 dorsal root ganglion neurons was about 12000. Seven days after axotomy, 24% of the dorsal root ganglion neurons were lost (P<0.001), and 54% were lost 28 days after axotomy (P<0.001). With regard to the percentage of peptide-expressing neurons, the results obtained showed that both galanin message-associated peptide (from <1% to about 21%) and neuropeptide Y (from <1% to about 16%) are upregulated, whereas calcitonin gene-related peptide is downregulated (from about 41% to about 14%) following axotomy. Results obtained with retrograde labeling of the axotomized dorsal root ganglion neurons indicate that the neuropeptide regulations may be even more pronounced, if the analysis is confined to the axotomized dorsal root ganglion neurons rather than including the entire neuron population. We also applied conventional profile-based counting methods to compare with the stereological data and, although the results were comparable considering the trends of changes following axotomy, the actual percentage obtained with the two methods differed markedly, both for neuropeptide Y- and, especially, for galanin message-associated peptide-positive neurons. These present results demonstrate that marked species differences exist with regard to the effect of nerve injury on dorsal root ganglion neurons. Thus, whereas no neuron loss is seen in rat up to 4 weeks after a 'mid-thigh' transection [Tandrup et al. (2000) J. Comp. Neurol. 422, 172-180], the present results indicate a dramatic loss already after 1 week in mouse. It is suggested that the proximity in physical distance of the lesion to the cell body is a critical factor for the survival of the target-deprived neurons. Finally, stereological methodology seems warranted when assessing the total number of neurons as well as changes in peptide regulations after axotomy in mouse.     

Effect of peripheral nerve cut on neuropeptides in dorsal root ganglia and the spinal cord of monkey with special reference to galanin

Summary Using the indirect immunofluorescence method andin situ hybridization, the localization and levels of immunoreactivities and mRNAs for several neuropeptides were studied in lumbar dorsal root ganglia and spinal cord of untreated monkeys (Macaca mulatto) and after unilateral transection of the sciatic nerve. Immunoreactive galanin, calcitonin gene-related peptide, substance P and somatostatin and their mRNAs were found in cell bodies in dorsal root ganglia of untreated monkeys and on the contralateral side of the monkeys with unilateral sciatic nerve lesion. After axotomy there was a marked decrease in the number of calcitonin gene-related peptide-, substance P- and somatostatin-positive neurons in dorsal root ganglia ipsilateral to the lesion, whereas the number of galanin positive cells strongly increased. A few neuropeptide tyrosine-positive cells were seen in after axotomy, whereas no such neurons were found in controls. No vasoactive intestinal polypeptide-, peptide histidine isoleucine-, cholecystokinin-, dynorphin-, enkephalin-, neurotensin-or thyrotrophin releasing hormone-positive cell bodies were seen in dorsal root ganglia of any of the groups studied. In the dorsal horn of the spinal cord all peptide immunoreactivities described above, except thyrotropin releasing hormone, were found in varying numbers of nerve fibres with a similar distribution in untreated monkeys and in the contralateral dorsal horn in monkey with unilateral sciatic nerve lesion. Two cholecystokinin antisera were used directed against the C- and N-terminal portions, respectively, showing a distinctly different distribution pattern in the dorsal horn. Somatostatin- and dynorphin-like immunoreactivities were also observed in small neurons in the dorsal horn. No certain effect of axotomy on these interneurons could be seen. However, marked changes were observed after this type of lesion for some peptide containing fibres in the ipsilateral dorsal horn. Thus, there was a marked increase in galanin-like immunoreactivity, whereas calcitonin gene-related peptide-, substance P-, somatostatin-, peptide histidine isoleucine neurotensin- and cholecystokinin-like immunoreactivities decreased. No changes could be observed in neuropeptide tyrosine or enkephalin-positive fibres. The present results demonstrate marked ganglionic and transganglionic changes in peptide levels after peripheral axotomy. When compared to published results on the effect of axotomy on peptides in dorsal root ganglia and spinal cord of rat, both similarities and differences were encountered. Thus, in contrast to rat there was no marked upregulation of vasoactive intestinal polypeptide/peptide histidine isoleucine or neuropeptide tyrosine after axotomy in the monkey, whereas galanin was increased in both species. Both in monkey and rat, calcitonin gene-related peptide, substance P and somatostatin decreased. The decrease in neurotensin, peptide histidine isoleucine, and genuine cholecystokinin seen in monkey after axotomy has not been reported in the rat. Experimental studies on rat suggest that galanin may be an endogenous analgesic compound, active particularly after peripheral nerve lesions. We have therefore recently proposed that galanin agonists may be used in treatment of chronic pain, and the present demonstration that galanin is regulated in a similar fashion in a primate gives further support to the proposal to test galanin as an analgesic in human.     

Nerve injury increases an excitatory action of neuropeptide Y and Y2-agonists on dorsal root ganglion neurons

Damage to sensory nerves invokes the expression of neuropeptide Y in the cell bodies of sensory neurons in dorsal root ganglia. We therefore compared the action of this peptide on control dorsal root ganglia neurons with its action on neurons from animals in which the sciatic nerve had been cut. Neuropeptide Y (0.1-1.0 microM) increased the excitability of 24% of control neurons and its effect was stronger and more cells (56%) were affected after axotomy. Increased excitability was mediated via a Y2-receptor and resulted from attenuation of Ca2+-sensitive K+-conductance(s) secondary to suppression of N-type Ca2+ channel current. Y1-agonists potentiated L-type Ca2+ channel current in control neurons without altering excitability. This Y1-effect was attenuated whereas effects mediated via Y2-receptors were enhanced after axotomy. No evidence was found for involvement of Y4- or Y5-receptor subtypes in the actions of neuropeptide Y either on control or on axotomized dorsal root ganglion neurons. It is concluded that neuropeptide Y increases the excitability of sensory neurons by interacting with a Y2-receptor and thereby decreasing N-type Ca2+ channel current and Ca2+-sensitive K+-conductance(s). When peripheral nerves are damaged, dorsal root ganglion neurons start to express neuropeptide Y and its excitatory Y2-excitatory effects are enhanced. The peptide may therefore contribute to the generation of aberrant sensory activity and perhaps to the etiology of injury-induced neuropathic pain.     

Some nerve endings in the rat pelvic paracervical autonomic ganglia and varicosities in the uterus contain calcitonin gene-related peptide and originate from dorsal root ganglia

The pelvic paracervical autonomic ganglia of female rats were studied for a subpopulation of nerve endings that could be derived from sensory nerve fibers. Immunohistochemical staining using an antiserum against the synaptic-terminal protein synapsin I was used to identify terminal boutons, while an antiserum against the neuropeptide calcitonin gene-related peptide was used to reveal a subpopulation of sensory nerve fibers. The uterine cervix was also examined for the existence of calcitonin gene-related peptide and synapsin I immunoreactivity in nerve fiber varicosities. In addition, the location of nerve endings in the paracervical ganglion was compared to that in the superior cervical ganglion. Synapsin I immunoreactivity was present in the paracervical ganglion in abundant boutons around neuron somata and in the cervix in varicose nerve fibers of the myometrium, vasculature and epithelium. Double labeling immunocytochemistry revealed calcitonin gene-related peptide-like immunoreactivity in subpopulations of synapsin I-immunoreactive endings in ganglia and nerve varicosities in the cervix. Injection of a retrograde axonal tracer, fluorogold, into the paracervical ganglion produced labeled neurons in dorsal root ganglia and spinal cord; however, fluorogold-labeled neurons containing calcitonin gene-related peptide immunoreactivity were visualized only in dorsal root ganglia. Injections of fluorogold into the uterine cervix produced labeled neurons in the paracervical ganglion and dorsal root ganglia; however, only those in dorsal root ganglia contained immunoreactivity for calcitonin gene-related peptide. These results suggest that immunoreactivity for calcitonin gene-related peptide is present in a subpopulation of nerve endings in the paracervical ganglion and not merely in fibers of passage. The nerve endings in the ganglion and varicosities in the uterine cervix originate from sensory neurons in dorsal root ganglia. The arrangement of endings in the ganglia could play a role in sensory/autonomic interactions for modulation of visceral activity.     

Neuropeptide expression in cultures of adult sensory neurons: modulation of substance P and calcitonin gene-related peptide levels by nerve growth factor

In contrast to developing sensory neurons, the survival of adult rat dorsal root ganglion neurons in pure neuronal culture is not dependent on specific neurotrophic factors such as nerve growth factor or brain-derived neurotrophic factor [Lindsay R. M. (1988) J. Neurosci. 8, 2394-2405]. In the present study we have examined possible modulatory effects of nerve growth factor on the neuropeptide content of sub-populations of adult rat dorsal root ganglion neurons in vitro. During the first 1-2 days in culture the neuropeptides substance P and calcitonin gene-related peptide could be detected by immunofluorescence staining in cultures grown in the presence or absence of nerve growth factor, but at longer times in nerve growth factor-deprived cultures there was loss of immunoreactive staining for both peptides. In the presence of nerve growth factor, however, the percentage of substance P- and calcitonin gene-related peptide-immunoreactive neurons remained relatively constant, for at least 14 days, at levels that were similar to the percentage of such peptide-containing neurons found in sections of adult rat dorsal root ganglia. Quantitation by radioimmunoassay of the levels of substance P and calcitonin gene-related peptide in cultures grown in the presence or absence of nerve growth factor agreed with the qualitative observations obtained by immunofluorescence: 10-15-fold higher levels of substance P and calcitonin gene-related peptide were found in cultures grown with nerve growth factor for 18 days, as compared to nerve growth factor-deprived cultures. In nerve growth factor-treated cultures increased levels of substance P and calcitonin gene-related peptide were observed within 3-6 days in vitro, and further steady increases in the levels of both peptides were found up to 18 days. A low basal level of both peptides could always be detected, even in the presence of an excess of antibodies to nerve growth factor. Up-regulation of the synthesis of substance P and calcitonin gene-related peptide did not depend on nerve growth factor being present at the initiation of the cultures, as elevated levels of both peptides could be induced in cultures even after up to 10 days' prior deprivation of nerve growth factor. Removal of nerve growth factor from the cultures resulted in reduced levels of peptide within 3 days.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphine treatment induced calcitonin gene-related peptide and substance P increases in cultured dorsal root ganglion neurons

The mechanism of spinal tolerance to the analgesic effects of opiates is unclear at present. We have reported previously that calcitonin gene-related peptide-like immunoreactivity was significantly increased in primary afferents of the spinal dorsal horn during the development of morphine tolerance, suggesting that changes in the level of pain-related neuropeptides in dorsal root ganglion neurons may be involved [Menard D. P. et al. (1996) J. Neurosci. 16, 2342-2351]. In this study, we investigated if in vitro treatment with morphine can mimic the in vivo findings and induce increases in calcitonin gene-related peptide-like immunostaining in cultured dorsal root ganglion neurons from young (three-month-old) and middle-aged (10-month-old) adult rats. Following a repetitive exposure to morphine sulfate (1, 5, 10 microM) for six days, the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons in cultured dorsal root ganglia from three- and 10-month-old rats was significantly increased. A lower concentration (0.5 microM) of morphine induced these increases only in dorsal root ganglion neurons from middle-aged rats. Morphine treatment was also found to increase the number of calcitonin gene-related peptide-immunoreactive neurons possessing multiple, long branches (i.e. with at least one branch >0.5mm). This apparent increase in the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons observed following morphine treatment was blocked by naloxone, an opiate antagonist, indicating the involvement of genuine opioid receptors. No significant change in the number of neuropeptide Y- or galanin-immunoreactive neurons in cultured dorsal root ganglia was detected following any of these treatments.These data suggest that repeated exposure to morphine rather selectively increases calcitonin gene-related peptide- and substance P-like immunoreactivity in cultured dorsal root ganglion neurons. Moreover, the sensitivity to morphine-induced changes is greater in cultured dorsal root ganglion neurons from 10- compared to three-month-old rats. Hence, cultured dorsal root ganglion neurons can provide a model to investigate the cellular and molecular mechanisms underlying alterations in neuropeptide levels following repeated exposure to opiates and their relevance to the development of opioid tolerance.     

Receptor binding sites for cholecystokinin, galanin, somatostatin, substance P and vasoactive intestinal polypeptide in sympathetic ganglia.

Sympathetic ganglia are innervated by neuropeptide-containing fibers originating from pre- and postganglionic sympathetic neurons, dorsal root ganglion neurons, and in some cases, myenteric neurons. In the present report receptor autoradiography was used to determine whether sympathetic ganglia express receptor binding sites for several of these neuropeptides including bombesin, calcitonin gene-related peptide-alpha, cholecystokinin, galanin, neurokinin A, somatostatin, substance P, and vasoactive intestinal polypeptide. The sympathetic ganglia examined included the rat and rabbit superior cervical ganglia and the rabbit superior mesenteric ganglion. High levels of receptor binding sites for cholecystokinin, galanin, somatostatin, substance P, and vasoactive intestinal polypeptide were observed in all sympathetic ganglia examined, although only discrete neuronal populations within each ganglion appeared to express receptor binding sites for any particular neuropeptide. These data suggest that discrete populations of postganglionic sympathetic neurons may be regulated by neuropeptides released from pre- and postganglionic sympathetic neurons, dorsal root ganglion neurons, and myenteric neurons.     

Transient expression of neuropeptide Y and its C-flanking peptide immunoreactivities in the spinal cord and ganglia of human embryos and fetuses.

An immunohistochemical study of spinal cord, dorsal root and sympathetic ganglia of human embryos and fetuses demonstrated that neuropeptide Y and its C-flanking peptide could be detected in seven-week-old embryos but were absent or difficult to demonstrate after the 17th week of gestation. The peptides were found in several structures of the spinal cord, e.g. fibres in the dorsal portion of the lateral funiculus, cell bodies and fibres in the dorsal horn, and motoneurons, and also in numerous primary sensory neurons of dorsal root ganglia. They were also present in sympathetic neurons and since these are the only structures expressing neuropeptide Y and its C-flanking peptide in the adult, it must be concluded that their presence in other neurons is a transient developmental feature. To assist in understanding the relationship of these transient structures with other spinal and sensory neurons, a comparison was made with other neuronal structures showing immunoreactivity for two general neuronal markers, neurofilaments and protein gene product 9.5, and two neuropeptides present in primary sensory afferents, somatostatin and substance P. In the dorsal root ganglia, numerous neuropeptide Y- and C-flanking peptide-immunoreactive neurons were observed before substance P- or somatostatin-immunoreactive cells could be detected. Therefore, neuropeptide Y and its C-flanking peptide could represent a primitive peptidergic system appearing before primary sensory neurons express their characteristic adult phenotype. The fibres of the lateral funiculus showing immunoreactivity for neuropeptide Y and its C-flanking peptide were longitudinally orientated and could be detected at all cephalocaudal levels of the spinal cord. Comparison with the other immunohistochemical markers indicated that they were not primary sensory afferents. At least some of them probably originated from neuropeptide Y- and C-flanking peptide-immunoreactive neurons of the dorsal horn, that may be considered to be a subset of early-appearing interneurons.     

Characterization of the peptidergic afferent innervation of the stomach in the rat, mouse and guinea-pig

Retrograde tracing of the fluorescent marker, True Blue, has been used together with immunohistochemistry employing antibodies to substance P, calcitonin gene-related peptide, somatostatin, vasoactive intestinal polypeptide and morphine-modulating peptide to study the afferent innervation of the stomach in rat, mouse and guinea-pig. Up to 85% of spinal afferents to the stomach in all three species contained immunoreactive calcitonin gene-related peptide, and up to 50% contained substance P. In all three species less than 10% of vagal afferents to the stomach reacted with antibodies to calcitonin gene-related peptide, or substance P. Cacitonin gene-related peptide-immunoreactive fibres were found in the myenteric plexus, circular muscle and around submucosal blood vessels in the stomach. In the rat, removal of the coeliac ganglion, splanchnic nerve section, or capsaicin treatment virtually abolished calcitonin gene-related peptide immunoreactivity in the stomach. Capsaicin and splanchnic section also abolished the staining of immunoreactive calcitonin gene-related peptide fibres in the coeliac ganglion. The same treatments abolished substance P staining of fibres around submucosal blood vessels, but in the myenteric plexus and circular smooth muscle there were still abundant immunoreactive fibres, presumably arising from intrinsic cell bodies. No somatostatin-containing visceral afferents could be found, although somatostatin was localized to cell bodies in rat dorsal root ganglia. Immunoreactive vasoactive intestinal polypeptide-containing dorsal root ganglia neurons were not found; although antibodies to morphine-modulatory peptide revealed immunoreactive nerve cell bodies, we were unable to exclude the possibility that this result is attributable to cross reactivity with calcitonin gene-related peptide. These results provide direct evidence that calcitonin gene-related peptide is a marker for a major subset of visceral primary afferent neurons and suggest that this population of spinal afferents makes a major contribution to the total gastric content of calcitonin gene-related peptide.     

Projections of peptide-containing neurons in rat colon

The distribution, origin and projections of nerve fibers containing vasoactive intestinal peptide, substance P, neuropeptide Y, galanin, gastrin-releasing peptide, calcitonin gene-related peptide, somatostatin or enkephalin were studied in the midcolon of the rat by immunocytochemistry and immunochemistry. Most of these nerve fibers had an intramural origin as was established by extrinsic denervation (serving of mesenterial nerves). Extrinsic denervation eliminated neuropeptide Y-containing fibers of presumably sympathetic origin together with sensory nerve fibers containing both substance P and calcitonin gene-related peptide. Co-existence of two peptides in the same neuron was studied by double immunostaining. This revealed co-existence of neuropeptide Y and vasoactive intestinal peptide in one population of intramural neurons; an additional population of intramural neurons was found to contain vasoactive intestinal peptide but not neuropeptide Y. All somatostatin-containing neurons in the submucous ganglia were found to harbor calcitonin gene-related peptide. A much larger population of submucous neurons containing calcitonin gene-related but not somatostatin was also detected. Some perivascular calcitonin gene-related peptide-containing nerve fibers (of intrinsic origin) harbored vasoactive intestinal peptide while others (of extrinsic origin) harbored substance P. The polarities and projections of the various peptide-containing intramural neurons in the transverse colon were studied by analysing the loss of nerve fibers upon local disruption of enteric nervous pathways (myectomy or intestinal clamping). Myenteric neurons containing vasoactive intestinal peptide, galanin, gastrin-releasing peptide, calcitonin gene-related peptide, somatostatin or vasoactive intestinal peptide/neuropeptide Y gave off 5-10-mm-long descending projections while those containing substance P or enkephalin issued approx. 5-mm-long ascending projections. Submucous neurons containing calcitonin gene-related peptide, somatostatin/calcitonin gene-related peptide or gastrin-releasing peptide issued both ascending (2-6 mm) and descending (2-6 mm) projections, those containing vasoactive intestinal peptide issued ascending (approx. 2 mm) projections, while those containing galanin or vasoactive intestinal peptide/neuropeptide Y lacked demonstrable oro-anal projections. Enkephalin-containing fibers could not be detected in the mucosa and the mucosal substance P-containing nerve fibers were too few to enable us to delineate their projections.     

神经肽Y/降钙素基因相关肽在腰椎间盘中的分布与共表达

背景：研究发现,神经肽Y与降钙素基因相关肽在交感神经节中有共存现象。 目的：观察神经肽Y/降钙素基因相关肽在正常腰椎间盘的共分布及在突出腰椎间盘髓核组织中的共表达。 方法：从10例尸体中收集完整的腰椎间盘,在另10例尸体中收集腰椎间盘髓核组织作为对照组。收集30例有症状的腰椎间盘突出症患者,其中L4/5或L5/S1需行腰椎间盘髓核摘除,取出髓核组织作为实验组。 结果与结论：①正常椎间盘组织中神经肽Y/降钙素基因相关肽荧光双标染色阳性的神经纤维较多的分布于椎间盘纤维环外1/3,但在椎间盘内2/3及髓核中未见或少量分布。②髓核的共表达,神经肽Y/降钙素基因相关肽荧光双标染色神经纤维的阳性率实验组均高于对照组(P < 0.05)。提示在正常腰椎间盘组织中神经肽Y/降钙素基因相关肽分布于纤维环外1/3部分,在纤维环内2/3部分及髓核组织未分布,但在突出椎间盘髓核组织中有大量的神经肽Y/降钙素基因相关肽共表达。     

Regulation of galanin and neuropeptide Y in dorsal root ganglia and dorsal horn in rat mononeuropathic models: possible relation to tactile hypersensitivity.

The expression of galanin and neuropeptide Y in rat lumbar 5 (L5) dorsal root ganglia and dorsal horn (L4-5) was studied after four types of peripheral nerve injury using immunohistochemistry and in situ hybridization. The possible correlation between these two peptides and tactile allodynia-like behaviour was analysed as well. The models employed were the Gazelius (photochemical lesion) and Seltzer and Bennett (constriction lesions) models, as well as complete sciatic nerve transection (axotomy). Two weeks after surgery, the Gazelius model rats more frequently displayed a greater tactile allodynia than the rats from the Seltzer and Bennett models. Tactile allodynia was not observed in any of the axotomized rats. A marked increase in the number of galanin-immunoreactive and galanin messenger RNA-positive neuron profiles was observed in ipsilateral dorsal root ganglia in all types of models. The increase in allodynic rats (Gazelius, Seltzer and Bennett models) was less pronounced than that after axotomy. In addition, in the Bennett model the number of galanin-immunoreactive neurons was significantly lower in allodynic rats as compared to non-allodynic rats, and the same tendency, but less obvious was found in the Seltzer model. Furthermore, an increase in galanin-immunoreactive fibres was found in the superficial laminae of the ipsilateral dorsal horn in all lesion models, especially in lamina II. A dramatic increase in the number of neuropeptide Y and neuropeptide Y messenger RNA-positive neuron profiles was also found in the ipsilateral dorsal root ganglia in all models, but no significant difference was found in peptide levels between allodynic and non-allodynic rats in any of the models. The present results suggest that the levels of endogenous galanin may play a role in whether or not allodynia develops in the Bennett model.     

Neuropeptide Y receptor-expressing dorsal horn neurons: Role in nocifensive reflex responses to heat and formalin

Lumbar intrathecal injection of neuropeptide Y (NPY) is antinociceptive, particularly in models of nerve injury and inflammation. Intrathecal NPY does not alter nociception in mice null for the Y1 neuropeptide Y receptor (Y1R) and these mice show enhanced nocifensive reflex responses to aversive thermal, mechanical, visceral and chemical stimuli. Y1R and NPY receptor type 2 (Y2R) are present in the spinal dorsal horn presynaptically on primary afferent, and possibly interneuron terminals, but only Y1R is found postsynaptically on dorsal horn neurons. In the present study, we sought to assess the anatomic effects of lumbar intrathecal disulfide conjugate of neuropeptide Y and saporin (NPY-sap) and to determine the role of Y1R-expressing dorsal horn neurons in nocifensive responses to aversive thermal and chemical stimulation. Lumbar intrathecal injection of NPY-sap was used to selectively destroy Y1R-expressing lumbar dorsal horn neurons followed by testing nocifensive reflex responses on the hotplate and after hind-paw formalin injection. NPY-saporin decreased superficial dorsal horn staining for Y1R, but not neurokinin-1 receptor, mu opiate receptor or NPY peptide, and had no effect on Y1R cell counts in fourth lumbar spinal segment dorsal root ganglia. Loss of Y1R-expressing dorsal horn neurons was associated with increased first response latencies on the 44 °C hotplate and reduced total time rats spent licking and guarding hind paws during 600 s trials at 44 °C or 200 s trials at 47 °C. First hind-paw response latencies to high intensity phasic stimulation at 52 °C were unaffected. NPY-sap also reduced formalin-induced nocifensive behaviors during both interphase and phase II. These data demonstrate that selective destruction of Y1R-expressing superficial dorsal horn neurons, probably excitatory interneurons and/or projection neurons, reduces nocifensive reflex responses, particularly to activation of C nociceptors, and suggest a possible role for Y1R-expressing dorsal horn neurons in pain.     

Regulation of neuronal death and calcitonin gene-related peptide by fibroblast growth factor-2 and FGFR3 after peripheral nerve injury: evidence from mouse mutants

The presence and regulation of basic fibroblast growth factor and its high-affinity tyrosine kinase receptor FGFR3 in sensory neurons during development and after peripheral nerve injury suggest a physiological role of the fibroblast growth factor-2 system for survival and maintenance of sensory neurons. Here we investigated L5 spinal ganglia of intact and lesioned fibroblast growth factor-2 knock-out and FGFR3 knock-out mice. Quantification of sensory neurons in intact L5 spinal ganglia revealed no differences between wild-types and mutant mice. After sciatic nerve axotomy, the normally occurring neuron loss in wild-type mice was significantly reduced in both knock-out strains suggesting that fibroblast growth factor-2 is involved in neuronal death mediated via FGFR3. In addition, the number of chromatolytic and eccentric cells was significantly increased in fibroblast growth factor-2 knock-out mice indicating a transient protection of injured spinal ganglia neurons in the absence of fibroblast growth factor-2. The expression of the neuropeptide calcitonin gene-related peptide in sensory neurons of intact fibroblast growth factor-2 knock-out and FGFR3 knock-out mice was not changed in comparison to adequate wild-types. Fibroblast growth factor-2 wild-type and FGFR3 wild-type mice showed a lesion-induced decrease of calcitonin gene-related peptide-positive neurons in ipsilateral L5 spinal ganglia whereas the loss of calcitonin gene-related peptide-immunoreactive sensory neurons is reduced in the absence of fibroblast growth factor-2 or FGFR3, respectively. In addition, FGFR3 wild-type and knock-out mice displayed a contralateral reduction of the neuropeptide after axotomy. These results suggest that endogenous fibroblast growth factor-2 and FGFR3 are crucially involved in the regulation of survival and calcitonin gene-related peptide expression of lumbar sensory neurons after lesion, but not during development.     

Expression of neuropeptides and neuropeptide mRNAs in spinal cord after axotomy in the rat,with special reference to motoneurons and galanin

The extent to which the plasticity in peptide expression observed in developing spinal motoneurons occurs following proximal peripheral axotomy in the adult rat was examined using in situ hybridization and immunohistochemical techniques to visualize the changes. Transient upregulation of galanin, vasoactive intestinal polypeptide (VIP) and substance P messenger ribonucleic acids (mRNAs) was observed within subpopulations of motoneurons ipsilateral to lesion for periods lasting 2–3 weeks after injury. In contrast, the axotomy-induced heterogenous increases in somatostatin and neuropeptide tyrosine mRNA expression in ipsilateral motoneurons remained elevated, or, in the case of somatostatin, continued to increase for the time period studied (1 month). Immunohistochemical analysis agreed with the in situ hybridization results, showing some motoneurons within the injured ventral horn to contain galanin-, VIP-or somatostatin-like immunoreactivity. In some instances, galanin-immunoreactive motoneurons colocalized with calcitonin gene-related peptide immunoreactivity. Most of the neurons expressing the injury-induced peptides appeared large, presumably alpha-motoneurons, but there were also many small neurons expressing galanin in the ventral horn ipsilateral to lesion. This may represent evidence for peptide synthesis in gamma-motoneurons. The only peptide mRNA studied to be downregulated in response to axotomy was enkephalin. The results show that peptide expression in injured motoneurons is dramatically altered, the significance of which remains to be determined.     

Comparative study on the innervation of nerves with calcitonin gene-related peptide, substance P and neurokinin A immunoreactivity in the walls of the cerebral arteries of small bats (Mammalia: Microchiroptera).

The distribution and origin of nerves with calcitonin gene-related peptide, substance P or neurokinin A immunoreactivity in the walls of the cerebral arteries were investigated in three microchiropteran species. The supply of nerves immunoreactive for substance P and neurokinin A to the bat cerebral arteries is confined mostly to the vertebral and basilar arteries. The density of innervation of calcitonin gene-related peptide-immunoreactive nerves and that of nerves with substance P or neurokinin A immunoreactivity in the vertebrobasilar system differ among species: the Japanese large footed bat is innervated with nerves with calcitonin gene-related peptide, substance P and neurokinin A immunoreactivity with about the same density, whereas in the greater horseshoe bat, there are many substance P-immunoreactive nerves with very weak or no calcitonin gene-related peptide immunoreactivity, and in the bent-winged bat, calcitonin gene-related peptide immunoreactivity is not found in all nerves with substance P immunoreactivity in the pial arteries of all parts of the brain. Nearly all cells immunoreactive for substance P, calcitonin gene-related peptide or both in the trigeminal and cervical dorsal root ganglia were small. In the greater horseshoe bat and the bent-winged bat, there is a correlation between the level of expression of substance P and calcitonin gene-related peptide immunoreactivity in the cervical dorsal root and trigeminal ganglia and the cerebral perivascular nerves supplying the vertebrobasilar system. The cerebrovascular innervation of nerves with substance P immunoreactivity in small bats is similar to that of cats and guinea-pigs in which the trigeminal ganglia have been destroyed. These observations, in addition to absence of nerve cells showing substance P or calcitonin gene-related peptide immunoreactivity in the pial artery and in the nerve bundles accompanying the extracranial internal carotid and vertebral arteries, suggest that substance P-immunoreactive nerves with or without immunoreactivity to calcitonin gene-related peptide in the vertebrobasilar system of small bats originate exclusively from the cervical dorsal root ganglia. The remaining fibres probably originate in the trigeminal ganglia.     

Increased content and transport of substance P and calcitonin gene-related peptide in sensory nerves innervating inflamed tissue: evidence for a regulatory function of nerve growth factor in vivo.

The responses of sensory neuropeptides during unilateral, Freund's adjuvant-induced, paw inflammation in the rat were examined. After five days of inflammation, the substance P and calcitonin gene-related peptide content in the sciatic nerve supplying the inflamed paw were increased by 60-75% when compared with the contralateral side. At this time-point, there was also a 30-40% increase in the substance P and calcitonin gene-related peptide content of the dorsal root ganglia (L4-L6), and a 40% increase in the calcitonin gene-related peptide content of the L4-L6 segments of the dorsal spinal cord on the inflammation side. In the dorsal root ganglia, calcitonin gene-related peptide content was also increased as early as 12 h and 48 h after induction of paw inflammation. On day 5 of inflammation, the axonal transport of both sensory neuropeptides towards the inflamed paw, as determined after sciatic nerve ligation, was also markedly increased as compared with the control side. Despite this increased transport, the amount of substance P and calcitonin gene-related peptide present in the inflamed paw itself was either reduced or remained unchanged from day 1 through to day 5 of inflammation pointing towards reduced storage and increased release of the peptides in the inflamed tissue. Nerve growth factor content was markedly increased in the sciatic nerve of the inflamed paw with a peak of +136% at time-point 24 h after induction of inflammation. When rats were systemically treated with anti-nerve growth factor serum, the increase in neuropeptide content in the sciatic nerve of the inflamed paw (day 5) was prevented. On the other hand, local injections of nerve growth factor for 5 days into a noninflamed paw were able to induce an increase in substance P and calcitonin gene-related peptide content in the supplying sciatic nerve. These findings point towards a regulatory function for nerve growth factor in vivo in the stimulation of sensory neuropeptide synthesis during prolonged inflammatory processes.     

Ontogeny of acetylcholinesterase, substance P and calcitonin gene-related peptide-like immunoreactivity in chick dorsal root ganglia.

The distribution of acetylcholinesterase and of two neuropeptide (substance P and calcitonin gene-related peptide) immunoreactivities has been investigated in sensory neurons of lumbosacral dorsal root ganglia during chick embryo development, combining immunolocalization of neuropeptides with simultaneous histochemical detection of acetylcholinesterase, in order to study co-localization of the two peptides and their relations with acetylcholinesterase. Acetylcholinesterase at E7 of development appears in only a few neurons, usually the larger ones located in the lateroventral region of the ganglia. As development proceeds the number of neurons and intensity of staining increase. Until E12-13 acetylcholinesterase positivity is limited to the region of the ganglion containing larger neurons. At later stages (E20) it spreads progressively, leading to staining of cells over the whole ganglion. Substance P-like immunoreactivity appears at E6 and for calcitonin gene-related peptide at E7. These immunoreactivities progressively increase with development, remaining limited to the small neuron compartment of the dorsomedial region of the ganglion. Immunoreactivity for both neuropeptides reaches a maximum around E10-13 and then declines. Using simultaneous double immunostaining, calcitonin gene-related peptide and substance P-like immunoreactivities are largely co-localized, although their distribution is not completely coincident. Neuropeptide-positive cells are usually devoid of any acetylcholinesterase activity until E15. They become positive for the enzyme at later stages. The significance of acetylcholinesterase expression in sensory neurons and the possible relation of its appearance and neuron size is discussed.     

Developmental pattern and distribution of nerve growth factor low-affinity receptor immunoreactivity in human spinal cord and dorsal root ganglia: comparison with synaptophysin, neurofilament and neuropeptide immunoreactivities.

Immunocytochemical expression of the low-affinity nerve growth factor receptor was studied in human fetal and adult tissues using the monoclonal antibody ME20.4. In dorsal root ganglia, a few immunoreactive neurons were first detected in nine-week-old fetuses and many more were found in the following weeks of gestation. However, none was present in adult ganglia. The ME20.4-positive cells were larger than neurons immunostained by substance P, calcitonin gene-related peptide or galanin antibodies. In the spinal cord, fibres immunostained by ME20.4 appeared in a characteristic pattern that differed from the spatial and temporal distributions of synaptophysin- and neurofilament-immunoreactive fibres. Those expressing the low-affinity nerve growth factor receptor were only detected in regions containing collaterals of primary sensory axons: (i) in the dorsal funiculus between seven and 18 weeks of gestation; (ii) in a ventrodorsal bundle reaching the ventral horn from weeks 12-14; (iii) in the medial region of the dorsal horn between weeks 12 and 20; (iv) in the superficial layers and lateral portion of the dorsal horn after the 14th week of gestation and also in adult spinal cord. During the fetal period, ME20.4 immunoreactivity was also found in motoneurons and peripheral nerve fibres in the skin, myotomes and gut. Sheaths of peripheral nerves and the adventitia of blood vessels were stained both in fetal and adult tissues. Thus, the low-affinity nerve growth factor receptor is: (i) strongly expressed in the developing human nervous system; (ii) transiently associated with a subset of large primary sensory neurons and with motoneurons; (iii) transiently and sequentially expressed by various groups of sensory afferents to the spinal cord; (iv) permanently expressed by fibres in the superficial layers of the dorsal horn, Clarke's column, nerve sheaths and the adventitia of blood vessels.