AMPA and NMDA glutamate receptors are found in both peptidergic and non-peptidergic primary afferent neurons in the rat

Two distinct classes of nociceptive primary afferents, peptidergic and non-peptidergic, respond similarly to acute noxious stimulation; however the peptidergic afferents are more likely to play a role in inflammatory pain, while the non-peptidergic afferents may be more characteristically involved in neuropathic pain. Using multiple immunofluorescence, we determined the proportions of neurons in the rat L4 dorsal root ganglion (DRG) that co-express AMPA or NMDA glutamate receptors and markers for the peptidergic and non-peptidergic classes of primary afferents, substance P and P2X(3), respectively. The fraction of DRG neurons immunostained for the NR1 subunit of the NMDA receptor (40%) was significantly higher than that of DRG neurons immunostained for the GluR2/3 (27%) or the GluR4 (34%) subunits of the AMPA receptor. Of all DRG neurons double-immunostained for glutamate receptor subunits and either marker for peptidergic and non-peptidergic afferents, a significantly larger proportion expressed GluR4 than GluR2/3 or NR1 and in a significantly larger proportion of P2X(3)- than SP-positive DRG neurons. These observations support the idea that nociceptors, involved primarily in the mediation of neuropathic pain, may be presynaptically modulated by GluR4-containing AMPA receptors.     

Hot receptors in the brain

Background

The complex neuronal circuitry of the dorsal horn of the spinal cord is as yet poorly understood. However, defining the circuits underlying the transmission of information from primary afferents to higher levels is critical to our understanding of sensory processing. In this study, we have examined phosphodiesterase 1C (Pde1c) BAC transgenic mice in which a green fluorescent protein (GFP) reporter gene reflects Pde1c expression in sensory neuron subpopulations in the dorsal root ganglia and spinal cord.

Results

Using double labeling immunofluorescence, we demonstrate GFP expression in specific subpopulations of primary sensory neurons and a distinct neuronal expression pattern within the spinal cord dorsal horn. In the dorsal root ganglia, their distribution is restricted to those subpopulations of primary sensory neurons that give rise to unmyelinated C fibers (neurofilament 200 negative). A small proportion of both non-peptidergic (IB4-binding) and peptidergic (CGRP immunoreactive) subclasses expressed GFP. However, GFP expression was more common in the non-peptidergic than the peptidergic subclass. GFP was also expressed in a subpopulation of the primary sensory neurons immunoreactive for the vanilloid receptor TRPV1 and the ATP-gated ion channel P2X₃. In the spinal cord dorsal horn, GFP positive neurons were largely restricted to lamina I and to a lesser extent lamina II, but surprisingly did not coexpress markers for key neuronal populations present in the superficial dorsal horn.

Conclusion

The expression of GFP in subclasses of nociceptors and also in dorsal horn regions densely innervated by nociceptors suggests that Pde1c marks a unique subpopulation of nociceptive sensory neurons.     

Topographically distinct epidermal nociceptive circuits revealed by axonal tracers targeted to Mrgprd

The brain receives sensory input from diverse peripheral tissues, including the skin, the body's largest sensory organ. Using genetically encoded axonal tracers expressed from the Mrgprd locus, we identify a subpopulation of nonpeptidergic, nociceptive neurons that project exclusively to the skin, and to no other peripheral tissue examined. Surprisingly, Mrgprd(+) innervation is restricted to the epidermis and absent from specialized sensory structures. Furthermore, Mrgprd(+) fibers terminate in a specific layer of the epidermis, the stratum granulosum. This termination zone is distinct from that innervated by most CGRP(+) neurons, revealing that peptidergic and nonpeptidergic epidermal innervation is spatially segregated. The central projections deriving from these distinct epidermal innervation zones terminate in adjacent laminae in the dorsal spinal cord. Thus, afferent input from different layers of the epidermis is conveyed by topographically segregated sensory circuits, suggesting that at least some aspects of sensory information processing may be organized along labeled lines.     

Changes in the immunoreactivity of substance P and calcitonin gene-related peptide in the laryngeal taste buds of chronically hypoxic rats

The distribution of substance P (SP)- and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the taste buds of the epiglottis and aryepiglottic folds was compared between normoxic control and chronically isocapnic hypoxic rats (10% O2 and 3-4% CO2 for 3 months). In the normoxic laryngeal taste buds, SP- and CGRP-immunoreactive fibers were detected within the taste buds, where they appeared as thin processes with many varicosities. Most CGRP fibers showed coexistence with SP, but a few fibers showed the immunoreactivity of CGRP only. The density of intra- and subgemmal SP and CGRP fibers penetrating into the laryngeal taste buds was significantly higher in chronically hypoxic rats than in normoxic control rats. Water intake in the hypoxic rats was significantly lower than in the normoxic rats. These results indicate that the increased density of SP- and CGRP-containing nerve fibers within the laryngeal taste buds is a predominant feature of hypoxic adaptation. The altered peptidergic innervation and reduced water intake support the hypothesis that the laryngeal taste buds are involved in water reception, and that the water reception may be under the control of peptidergic innervation.     

Capsaicin-sensitive afferents and their role in gastroprotection: an update

The pivotal role of capsaicin-sensitive peptidergic sensory fibers in the maintenance of gastric mucosal integrity against injurious interventions was suggested by the authors 20 years ago. Since then substantial evidence has accumulated for the local sensory-efferent function of the released CGRP, tachykinins and NO in this gastroprotective mechanism. This overview outlines some recent achievements which shed light on new aspects and further horizons in this field. (1) Cloning the capsaicin VR-1 receptor (an ion channel-coupled receptor) and raising the VR-1 knockout mice provided a definite molecular background for the existence of capsaicin-sensitive afferents with both sensory and mediator releasing functions in the stomach. This cation channel is also sensitive to hydrogen ions. (2) VR-1 agonists (capsaicin, resiniferatoxin, piperine) protect against gastric ulcer of the rat parallel with their sensory stimulating potencies. (3) Antidromic stimulation of capsaicin-sensitive vagal and somatic afferents results in the release of CGRP, tachykinins, NO and somatostatin. Somatostatin with gastroprotective effect is released from D cells and sensory nerve endings. (4) The recent theory for the existence of spinal afferents without sensory function [P. Holzer, C.A. Maggi, Dissociation of dorsal root ganglion neurons into afferent and efferent-like neurons, Neuroscience 86 (1998) 389-398] is discussed. Data proposed to support this theory are interpreted here on the basis of a dual sensory-efferent function of VR-1 positive afferents, characterized by a frequency optimum of discharges for release vasodilatory neuropeptides below the nociceptive threshold. (5) Recent data on the effect of capsaicin in healthy human stomach are summarized. These results indicate that the gastroprotective effect of capsaicin in the human stomach involves additional mechanisms to those already revealed in the rat.     

神经激肽-1(NK-1)受体介导福尔马林诱发的大鼠脊髓c-fos表达

本文用Ｆｏｓ作为背角伤害性反应神经元活动的一个标志物,结合免疫细胞化学和神经药理学方法,观察了速激肽受体拮抗剂对福尔马林诱发的脊髓ｃ－ｆｏｓ原癌基因表达的影响。一侧大鼠后肢跖部皮下注射福尔马林,仅在同侧脊髓背角有ｃ－ｆｏｓ表达。Ｆｏｓ阳性神经元最密集分布于Ｉ层和Ⅱ层背侧的内侧部,中等量分布于Ⅳ层和Ｖ型,少量定位于Ⅱ层腹侧部、Ⅲ、Ⅵ和Ⅹ层。若预先在一侧大鼠后肢跖部皮下注射福尔马林前,鞘内给予神经激肽     

Distribution and immunocytochemical characterization of dorsal root ganglion neurons innervating the lumbar intervertebral disc in rats: a review

Previously, it was believed that the lumbar intervertebral disc was innervated segmentally by dorsal root ganglion (DRG) neurons via the sinuvertebral nerves. Recently, it was demonstrated using retrograde tracing methods that the lower disc (L5-L6) is innervated predominantly by upper (L1 and L2) DRG neurons via the sympathetic trunks. Furthermore, we investigated the expression of various pain-related molecules such as calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), P2X(3) receptor and vanniloid receptor 1 (VR1) in DRG neurons innervating the disc using a combination of immunostaining with the retrograde tracing method. This review outlines the distribution and immunocytochemical characterization of DRG neurons innervating the disc. Small nociceptive DRG neurons are classified into nerve growth factor (NGF)-dependent neurons and glial cell line-derived neurotrophic factor (GDNF)-dependent neurons and they can be distinguished by their reactivity for CGRP and IB4, respectively. We found that about half of the neurons innervating the disc were CGRP-immunoreactive (-ir), whilst, only 0.6% of the DRG neurons were IB4-positive, thereby indicating that NGF-dependent neurons are the main subpopulation which transmits and modulates nociceptive information from the disc. In addition, we also demonstrated P2X(3)- and VR1-immunoreactivity in DRG neurons innervating the disc and noted that they were mainly localized in NGF-dependent neurons. It is well known that NGF has sensitizing effects on DRG neurons, with a recent study demonstratng the presence of NGF in the painful intervertebral disc. Therefore, it is suggested that NGF is involved in the generation of discogenic low back pain.     

Distribution of immunoreactive neuropeptides in the pancreas of the bullfrog,Rana catesbeiana,demonstrated by immunofluorescence

Indirect double immunofluorescence labelling for eight neuropeptides in the pancreas of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of certain neuropeptides in the exocrine and endocrine pancreas. Immunoreactivity of substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), FMRFamide (FMRF), and galanin (GAL) was localized in nerve fibers distributed between the acini and around the duct system and vasculature of the exocrine pancreas. In these regions, CGRP-immunoreactive fibers were more numerous than those containing the other five peptides. Almost all SP fibers showed coexistence of SP with CGRP, and about one third of fibers also showed coexistence of SP with VIP, NPY, FMRF, and GAL. In the endocrine pancreas, SP, CGRP, VIP, and GAL were recognized in the nerve fibers around and within the islets of Langerhans, and VIP and GAL fibers were more numerous than SP and CGRP fibers. All CGRP fibers, and about half of the VIP and GAL fibers were immunoreactive for SP. NPY- and FMRF-immunoreactive cells were found at the periphery of the islets. These findings suggest that the exocrine and endocrine pancreatic functions of the bullfrog are under the control of peptidergic innervation.     

Neuropeptide (neuropeptide Y, neurotensin, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, somatostatin) immunohistochemistry and ultrastructure of renal nerves

With the use of several region-specific antisera and the peroxidase-antiperoxidase (PAP) technique, several regulatory polypeptides were localized in nerves of the kidney. Neuropeptide Y (NPY)- immunoreactivity (IR), neurotensin (NT)-IR and vasoactive intestinal polypeptide (VIP)-IR occurred at high densities in all segments of the renal arterial system forming a perivascular plexus. Furthermore, NT-IR nerves were particularly frequent at the juxtaglomerular apparatus (JGA). Calcitonin gene-related peptide (CGRP)-IR was mainly concentrated in nerves supplying the hilus arteries and the JGA. Substance P (SP)-IR was predominantly found in large varicosities close to large renal arterial vessels and in the vicinity of the JGA. Somatostatin (SOM)-IR was only observed in single varicosities located at the media-adventitia border of large renal hilus arteries. The peptidergic nerves are correlated to their ultrastructural counterpart. In addition, the distribution patterns and the frequency of the different types of renal peptidergic nerve fibres are evaluated and compared. The functional role of these neuropeptides and their origin within the efferent branch of this part of the peripheral autonomic nervous system is discussed. Furthermore, the implication of some of the neuropeptides studied in afferent renal innervation is also substantiated.     

Neuropeptide distribution in the cervico-thoracic paravertebral ganglia of the cat with particular reference to calcitonin gene-related peptide immunoreactivity

Summary Paraffin sections of cervical and upper thoracic paravertebral ganglia of the cat were investigated by immunohistochemistry using antisera directed against calcitonin gene-related peptide (CGRP). The relationships of CGRP-immunoreactive structures to those exhibiting immunoreactivity to antisera against other regulatory peptides and dopamine--hydroxylase (DBH), respectively, were studied in consecutive sections. Singly scattered CGRP-immunoreactive neuronal perikarya were observed in the superior and middle cervical ganglia as well as in the stellate ganglion. These neurons also displayed immunoreactivity to vasoactive intestinal polypeptide (VIP), and some additionally exhibited faint substance-P immunoreactivity. DBH- and neuropeptide Y-immunoreactive ganglion cells were not identical with CGRP-immunoreactive neuronal cell bodies.According to the immunoreactive properties of varicosities, which abut on CGRP/VIP-immunoreactive perikarya, three types of CGRP/VIP-immunoreactive ganglion cells could be distinguished: (1) CGRP/VIP-immunoreactive neurons being surrounded by somatostatin-immunoreactive nerve fibers, (2) neurons being approached by both DBH- and met-enkephalin-immunoreactive varicosities, and (3) neurons receiving both DBH- and neurotensin-immunoreactive fibers. The stellate and upper thoracic ganglia harbored clusters of intensely VIP-immunoreactive somata, which lacked CGRP-immunoreactivity. Fine somatostatin-immunoreactive and coarse CGRP-immunoreactive fibers were distributed within these clusters, whereas patches of neurotensin-immunoreactive fibers were complementarily arranged. At all segmental levels investigated, a few postganglionic neurons were approached by both CGRP-immunoreactive and substance P-immunoreactive varicosities, but lacked a VIP-immunoreactive innervation. Therefore, CGRP/substance P-immunoreactive fiber baskets appeared rather to be of extraganglionic origin than to emerge from intraganglionic CGRP/VIP/SP neurons. CGRP-immunoreactive cell bodies or fibers were absent in clusters of small paraganglionic cells, but some of the solitary paraganglionic cells displayed CGRP-immunoreactivity. Our findings establish the presence of CGRP-immunoreactivity in a population of sympathetic neurons in the cat. A highly differentiated, segment-dependent organizational pattern of neuropeptides in cervico-thoracic paravertebral ganglia was demonstrated.Supported by Deutsche Forschungsgemeinschaft grant He 919/6-2     

Presynaptic functional trkB receptors mediate the release of excitatory neurotransmitters from primary afferent terminals in lamina II (substantia gelatinosa) of postnatal rat spinal cord

A subset of primary sensory neurons produces BDNF, which is implicated in control of nociceptive neurotransmission. We previously localized full-length trkB receptors on their terminals within lamina II. To functionally study these receptors, we here employed patch-clamp recordings, calcium imaging and immunocytochemistry on slices from 8-12 days post-natal rats. In this preparation, BDNF (100-500 ng/mL) enhances the release of sensory neurotransmitters (glutamate, substance P, CGRP) in lamina II by acting on trkB receptors expressed by primary afferent fibers of the peptidergic nociceptive type (PN-PAFs). Effect was blocked by trk antagonist K252a or anti-trkB antibody clone 47. A pre-synaptic mechanism was demonstrated after (i) patch-clamp recordings where the neurotrophin induced a significant increase in frequency, but not amplitude, of AMPA-mediated mEPSCs, (ii) real time calcium imaging, where sustained application of BDNF evoked an intense response in up to 57% lamina II neurons with a significant frequency rise. Antagonists of ionotropic glutamate receptors and NK(1) receptors completely inhibited the calcium response to BDNF. Reduction of CGRP (a specific marker of PN-PAFs) and substance P content in dorsal horn following BDNF preincubation, and analysis of the calcium response after depletion with capsaicin, confirmed that the neurotrophin presynaptically enhanced neurotransmitter release from PN-PAFs. This is the first demonstration that trkB receptors expressed by PN-PAF terminals in lamina II are functional during postnatal development. Implications of this finding are discussed considering that BDNF can be released by these same terminals and microglia, a fraction of which (as shown here) contains BDNF also in unactivated state.     

Neuropeptide (neuropeptide Y,neurotensin, vasoactive intestinal polypeptide,substance P,calcitonin gene-related peptide,somatostatin) immunohistochemistry and ultrastructure of renal nerves

Summary With the use of several region-specific antisera and the peroxidase-antiperoxidase (PAP) technique, several regulatory polypeptides were localized in nerves of the kidney. Neuropeptide Y (NPY)- immunoreactivity (IR), neurotensin (NT)-IR and vasoactive intestinal polypeptide (VIP)-IR occurred at high densities in all segments of the renal arterial system forming a perivascular plexus. Furthermore, NT-IR nerves were particularly frequent at the juxtaglomerular apparatus (JGA). Calcitonin gene-related peptide (CGRP)-IR was mainly concentrated in nerves supplying the hilus arteries and the JGA. Substance P (SP)-IR was predominantly found in large varicosities close to large renal arterial vessels and in the vicinity of the JGA. Somatostatin (SOM)-IR was only observed in single varicosities located at the media-adventitia border of large renal hilus arteries. The peptidergic nerves are correlated to their ultrastructural counterpart. In addition, the distribution patterns and the frequency of the different types of renal peptidergic nerve fibres are evaluated and compared. The functional role of these neuropeptides and their origin within the efferent branch of this part of the peripheral autonomic nervous system is discussed. Furthermore, the implication of some of the neuropeptides studied in afferent renal innervation is also substantiated.Dedicated to Prof. Dr. T.H. Schiebler on the occasion of his 65th birthday     

Distribution and ontogeny of SP, CGRP, SOM, and VIP in chick sensory and sympathetic ganglia

The distribution and ontogeny of four neuropeptides in developing chick lumbosacral sensory and sympathetic ganglia were studied using immunohistochemical techniques. Antibodies to two of these peptides, substance P (SP) and calcitonin gene-related peptide (CGRP), stained small neurons in the medial part of the dorsal root ganglia from embryonic Day 5 and Day 10, respectively, whereas neurons in the lateral part of the ganglia were negative; this distribution persisted throughout development. Both sets of neurons apparently send fibers to the dorsal horn of the spinal cord: SP to laminae I and II, and CGRP to lamina I, suggesting that the SP- and CGRP-positive sensory neurons are nociceptive or thermoreceptive. This correlation between the presence of SP or CGRP in a neuron and a particular functional modality thus provides evidence for a functional distinction between the mediodorsal and ventrolateral zones that are apparent during the development of chick dorsal root ganglia. Moreover, this study suggests that the type of neuron that develops within the dorsal root ganglion correlates with its position within the ganglion. In contrast to SP and CGRP, somatostatin (SOM) and vasoactive intestinal polypeptide (VIP) immunoreactivities were not seen in the lumbosacral sensory ganglia at any stage during development. However, both were present in sympathetic ganglia: SOM from embryonic Day 4.5 and VIP from embryonic Day 10. VIP immunoreactivity persisted throughout development in a large number of sympathetic neurons, but the number of cells with SOM immunoreactivity decreased from embryonic Day 10 onward. SOM therefore appears to be present only transiently in most chick lumbosacral sympathetic cells.     

Prostatic Acid Phosphatase Is Expressed in Peptidergic and Nonpeptidergic Nociceptive Neurons of Mice and Rats

Thiamine monophosphatase (TMPase, also known as Fluoride-resistant acid phosphatase or FRAP) is a classic histochemical marker of small- to medium-diameter dorsal root ganglia (DRG) neurons and has primarily been studied in the rat. Previously, we found that TMPase was molecularly identical to Prostatic acid phosphatase (PAP) using mice. In addition, PAP was expressed in a majority of nonpeptidergic, isolectin B4-binding (IB4⁺) nociceptive neurons and a subset of peptidergic, calcitonin gene-related peptide-containing (CGRP⁺) nociceptive neurons. At the time, we were unable to determine if PAP was present in rat DRG neurons because the antibody we used did not cross-react with PAP in rat tissues. In our present study, we generated a chicken polyclonal antibody against the secretory isoform of mouse PAP. This antibody detects mouse, rat and human PAP protein on western blots. Additionally, this antibody detects PAP in mouse and rat small- to medium-diameter DRG neurons and axon terminals in lamina II of spinal cord. In the rat, 92.5% of all PAP⁺ cells bind the nonpeptidergic marker IB4 and 31.8% of all PAP⁺ cells contain the peptidergic marker CGRP. Although PAP is found in peptidergic and nonpeptidergic neurons of mice and rats, the percentage of PAP⁺ neurons that express these markers differs between species. Moreover, PAP⁺ axon terminals in the rat partially overlap with Protein kinase Cγ (PKCγ⁺) interneurons in dorsal spinal cord whereas PAP⁺ axon terminals in the mouse terminate dorsal to PKCγ⁺ interneurons. Collectively, our studies highlight similarities and differences in PAP localization within nociceptive neurons of mice and rats.     

TH-, NPY-, SP-, and CGRP-immunoreactive nerves in interscapular brown adipose tissue of adult rats acclimated at different temperatures: an immunohistochemical study

Interscapular brown adipose tissue (IBAT), a site of nonshivering thermogenesis in mammals, is neurally controlled. The co-existence of sympathetic and peptidergic innervation has been demonstrated in different brown adipose depots. We studied the morphological profile of IBAT innervation and tested by immunohistochemical methods whether cold and warm stimulation are accompanied by modifications in the density of parenchymal noradrenergic nerve fibers. We also studied the immunoreactivity of afferent fibers—which contain calcitonin gene-related peptide (CGRP) and substance P (SP)<197>in different functional conditions. IBAT was obtained from adult rats (6 weeks old) acclimated at different temperatures (4°, 20°, and 28°C). Tissue activity was evaluated by studying the immunolocalization of uncoupling protein (UCP-1), a specific marker of brown adipose tissue. Noradrenergic and peptidergic innervation were seen to arise from morphologically different nerves. Fibers staining for tyrosine hydroxylase (TH) were thin, unmyelinated hilar nerves, and CGRP- and SP-positive fibers were in thick nerves containing both myelinated and unmyelinated fibers. Under cold stimulation, noradrenergic neurons produce greater amounts of TH, and their axons branch, resulting in increased parenchymal nerve fibers density. Neuropeptide Y (NPY) probably co-localizes with TH in noradrenergic neurons, but only in the perivascular nerve fiber network. The parenchymal distribution of NPY to interlobular arterioles and capillaries suggests that this peptide must have other functions besides that of innervating arteriovenous anastomoses, as hypothesized by other researchers. The different distribution of CGRP and SP suggests the existence of different sensory neuronal populations. The detection of CGRP at the parenchymal level is in line with the hypothesis of a trophic action of this peptide.     

去甲肾上腺素在脊髓背角的镇痛机制

疹髓背角浅层是传递和调制外周伤害性信息的关键部位。起源于脑干的去甲肾上腺素（NA）能纤维终止脊髓背角,它们释放的NA具有抑制初级传入末梢释放谷氨酸和P物质、增加Ⅱ层（胶状质）抑制性神经活性物质释放的作用。此外,形态学研究提示NA可能直接抑制Ⅰ/Ⅲ层向丘脑传递伤害性信息的投射神经元。NA可能通过以上途径,实现对外周伤害性信息传递的调制而发挥镇痛作用。     

A simultaneous demonstration of particular enteric neuronal cell types with the NADH:nitro BT-dehydrogenase reaction and of nerve fibres containing enkephalin-like immunoreactivity in the myenteric plexus of the porcine small intestine

The myenteric plexus of the porcine small intestine is studied using a combined method for the simultaneous visualization of enteric intramural neuronal cell bodies and peptidergic nerve fibers. As earlier reported, the histochemical method for demonstration of the NADH-dependent dehydrogenase reaction allows the identification of the three neuron types of Dogiel but, in addition, the afore mentioned staining method creates fair conditions in the tissue for the subsequent indirect immunocytochemical visualization of neuropeptides, as demonstrated in this work by means of the indirect immunofluorescence method for enkephalin-like immunoreactivity. Intense fluorescent varicosities of enkephalin-like nerve fibres were found to ramify around dark-blue stained ganglionic cells of type I, type II and type III in a manner suggestive of innervation.     

Distribution of five peptides, three general neuroendocrine markers, and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: an immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I-II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I-III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I-II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.     

Effect of intestinal inflammation on capsaicin-sensitive afferents in the ileum of<Emphasis Type="Italic"> Schistosoma mansoni</Emphasis>-infected mice

In the present study, the effect of intestinal schistosomiasis on the extrinsic sensory innervation of the murine ileum was investigated. Immunocytochemical techniques to localize calcitonin gene-related peptide (CGRP), substance P (SP), and vanilloid receptor 1 (VR1) were combined with retrograde tracing techniques and capsaicin treatment. Neurochemical characterization of extrinsic primary afferent neurons (EPANs) in normal and capsaicin-treated mice, revealed that CGRP and VR1, but not SP, were expressed in extrinsic afferents. Immunocytochemical analysis using the above-mentioned antibodies yielded three different populations of neurons in both dorsal root and nodose ganglia, namely CGRP/--, SP/--, and CGRP/SP-expressing neurons. Retrograde tracing revealed that only CGRP/--expressing neurons projected to the ileum. Intestinal schistosomiasis resulted in an upregulation of the number of CGRP-immunoreactive (ir) nerve fibers in the lamina propria of the villi, coinciding with an increase in mucosal mast cells in acutely and chronically infected animals. In infected animals, mucosal mast cells were found closely associated with a dense mucosal CGRP-ir fiber network. Neonatal capsaicin treatment led to a 70% reduction in the number of mucosal mast cells. In conclusion, the present study provides evidence that CGRP is a valid marker for EPANs in the mouse ileum, which are involved in the recruitment of mucosal mast cells. Morphological evidence is provided of a neuroimmune interaction between mucosal mast cells and EPANs in schistosoma-infected mice.     

Fine structural analysis of calcitonin gene-related peptide in the mouse inferior olivary complex

Climbing fiber afferents to the cerebellum, from the inferior olivary complex, have a powerful excitatory effect on Purkinje cells. Changes in the responsiveness of olivary neurons to their afferent inputs, leading to changes in the firing rate or pattern of activation in climbing fibers, have a significant effect on the activation of cerebellar neurons and ultimately on cerebellar function. Several neuropeptides have been localized in both varicosities and cell bodies of the mouse inferior olivary complex, one of which, calcitonin gene related peptide (CGRP), has been shown to modulate the activity of olivary neurons. The purpose of the present study was to investigate the synaptic relationships of CGRP-containing components of the caudal medial accessory olive and the principal olive of adult mice, using immunohistochemistry and electron microscopy. The vast majority of immunoreactive profiles were dendrites and dendritic spines within and outside the glial boundaries of synaptic glomeruli (clusters). Both received synaptic inputs from non-CGRP labeled axon terminals. CGRP was also present within the somata of olivary neurons as well as in profiles that had cytological characteristics of axons, some of which were filled with synaptic vesicles. These swellings infrequently formed synaptic contacts. At the LM level, few, if any, CGRP-immunoreactive climbing fibers, were seen, suggesting that CGRP is compartmentalized within the somata and dendrites of olivary neurons and is not transported to their axon terminals. Thus, in addition to previously identified extrinsic sources of CGRP, the widespread distribution of CGRP within olivary somata and dendrites identifies an intrinsic source of the peptide suggesting the possibility of dendritic release and a subsequent autocrine or paracrine function for this peptide within olivary circuits.