Containing Calcitonin Gene-related Peptide Research Articles

Chemically distinct preganglionic inputs to iris-projecting postganglionic neurons in the rat: A light and electron microscopic study

Individual autonomic postganglionic neurons are surrounded by pericellular baskets of preganglionic terminals that are easily identifiable with the light microscope. It has been assumed that the target cell of a pericellular basket of preganglionic terminals is the neuron at the centre of the basket. This assumption has enabled the connectivity of preganglionic neurons to be determined at the light microscopic level. However, if the preganglionic terminals in a pericellular basket make synapses with the dendrites of nearby, but functionally different, postganglionic neurons, then the conclusions of light microscopic studies are far less certain. We have used a serial section ultrastructural study to determine the target of the preganglionic pericellular basket in a situation where the apparent target cell is surrounded by neurons of dissimilar function. In the rat superior cervical ganglion, postganglionic neurons projecting to the iris were identified, using retrograde tracers, as single neurons (i.e., not in clusters). We have used immunohistochemistry to show that iris-projecting neurons are surrounded by preganglionic nerve terminals containing calcitonin gene-related peptide (CGRP). We have demonstrated that the pericellular basket of CGRP-immunoreactive preganglionic terminals provides inputs only to the soma at the centre of the basket and not to the dendrites of surrounding neurons. This suggests that, in autonomic ganglia, light microscopic identification of the preganglionic terminal baskets is likely to be a reliable method for identifying the targets of subclasses of preganglionic neurons.

Axotomy results in major changes in BDNF expression by dorsal root ganglion cells: BDNF expression in large trkB and trkC cells, in pericellular baskets, and in projections to deep dorsal horn and dorsal column nuclei.

Brain derived neurotrophic factor (BDNF) is normally expressed by a small number of predominantly trkA-expressing dorsal root ganglion cells. Using immunocytochemistry and in situ hybridization, we have examined the effect of sciatic nerve section on the expression of BDNF in the adult rat. Following axotomy there was a long lasting (4-week) increase in BDNF mRNA and protein in large-diameter, trkB- and trkC-expressing dorsal root ganglion cells. By 2 days postaxotomy, expression of BDNF mRNA had increased from 2% of trkB cells to 50%, and from 18% of trkC cells to 56%. In contrast, BDNF expression in most trkA cells was unchanged, although was increased in the small population of medium- and large-sized trkA cells. Following axotomy, BDNF-immunoreactive terminals appeared in the central axonal projections of large-diameter cells, including the deep dorsal horn and gracile nucleus. Neuropeptide Y was also upregulated following axotomy and was coexpressed with BDNF in the cell bodies and central terminals of the large cells. Ultrastructural analysis in lamina IV of the spinal cord revealed that BDNF terminals in these central projections establish synaptic contacts. Immunoreactivity at 4 weeks was also observed in pericellular baskets that contained calcitonin gene-related peptide (CGRP) and surrounded trkA- and trkB-expressing cells in L4 and L5 lumbar ganglia. These baskets are likely to arise from local, highly immunoreactive, BDNF/CGRP/trkA-expressing cells. Our results identify several novel targets for BDNF and imply that it acts locally in both autocrine and paracrine modes, as well as centrally in a synaptic mode, to modulate the response of somatosensory pathways in nerve injury.

Enhanced Sensory Reinnervation of Dental Target Tissues in Rats Following Low Level Laser (LLL) Irradiation

. Previous studies have suggested that low level laser (LLL) treatment at specific wavelengths can enhance motor and sensory function in peripheral nerves after injury. The dental pulp is innervated by unmyelinated C fibres and small myelinated Aδ fibres derived from the trigeminal ganglion, both of which are known to contain calcitonin gene related peptide (CGRP). The aim of the present study was to examine the effects of daily LLL treatment on the sensory reinnervation of the first molar pulp 10 days subsequent to inferior alveolar nerve (IAN) axotomy, by counting the number of nerve fibre profiles immunoreactive to CGRP in rats. Axotomy of the IAN was performed via an extraoral buccal incision. The animals (n=11) were divided into two groups receiving either daily LLL treatment with a GaAlAs laser (λ=830 nm), or sham laser treatment over the site of injury for 10 days postoperatively. The animals were transcardially perfused and fixed at death before excising the jaws for further fixation and demineralisation. The jaws were cryosectioned obliquely through the mesial root pulp of the first molar tooth, which was the chosen level for evaluation of reinnervation. The CGRP antigen–antibody binding sites were visualised using a standard avidin biotin peroxidase technique. Both sham and LLL treatment and the evaluation of results were conducted blind. The results were statistically analysed and presented as median with 25%–75% quartiles. A statistically significant (p≤0.0000) greater number of CGRP immunoreactive nerve profiles were seen in the LLL-treated group (median=5, range 4–6) compared to the sham laser treated group (median=2, range 1–3), at the specified area for evaluation. These results suggest that LLL treatment can enhance reinnervation of denervated dental tissues after IAN axotomy in the rat. The enhanced reinnervation could be due to accelerated regeneration of the axotomised nerve, to collateral reinnervation, or a combination of both these nerve responses. The mechanisms whereby these changes are effected are still unknown.

Pulmonary neuroendocrine cells in nitrofen-induced diaphragmatic hernia and the effect of prenatal glucocorticoids.

The high mortality associated with congenital diaphragmatic hernia (CDH) is due to pulmonary hypoplasia and hypertension, structural and functional abnormalities which can to some extent be ameliorated by prenatal administration of glucocorticoids. In the hypoplastic, hypertensive lungs of neonatal rats in which CDH has been induced by nitrofen, those pulmonary neuroendocrine cells (PNCs) containing calcitonin gene-related peptide (CGRP) increase in number, and it has been suggested that this might be due to inhibition of secretion of the peptide, the consequent decrease in its vasodilatory effects contributing to the hypertension. Whether this increase affects the entire population of PNCs, however, and how these cells are affected by administration of prenatal glucocorticoids, is unknown. As revealed by immunolabelling for protein gene product (PGP) 9.5, a general marker of NCs and expressed per cm2 tissue section, the total PNC population in rats with nitrofen-induced CDH was significantly greater than in controls receiving only olive oil (672 vs 375/cm2, P = 0.03) and was further increased (824 per cm2) in animals treated prenatally with dexamethasone (n = 8 in all groups). The increase in the total PNC population in rats with CDH is similar in magnitude to that described for the CGRP-containing subpopulation. Since the major role of the products of PNCs is now thought to be the regulation of development of pulmonary tissues and their response to injury, it is probable that the expansion of their population in the abnormal lungs associated with CDH is an adaptive response to pulmonary maldevelopment, a response possibly augmented by exogenous corticosteroids.

Patterns of innervation of sympathetic vascular neurons by peptide-containing primary sensory fibers

The purpose of this study was to determine whether there is a specific organization of the primary sensory innervation on to identified vascular neurons in the inferior mesenteric ganglion (IMG) in guinea-pig. Retrograde tracers were placed intraluminally in inferior mesenteric artery (IMA) or inferior mesenteric vein (IMV) in vitro to identify ganglionic neurons as arterial, venous or unlabeled neurons. The distribution of primary sensory nerve fibers containing calcitonin gene-related peptide (CGRP), neuronal nitric oxide synthase (NOS) and substance P immunoreactivity (SP-IR) was compared before and after treatment with capsaicin. In control animals the density of immunoreactivity varied both with the transmitter and the type of neuron innervated. The density of immunoreactivity for all the three substances was reduced by capsaicin treatment. The degree of reduction of immunoreactivity in the fibers varied with the transmitter and the type of neuron. The density of CGRP and SP immunoreactive fibers was greatest around unlabeled neurons; 78% of the CGRP fibers were of primary sensory origin and all of the SP fibers were primary sensory. Around arterial neurons 44% of the CGRP fibers were of primary sensory origin and around venous 68% were primary sensory. NOS positive innervation around venous neurons was denser than around arterial neurons and all of it was completely (97%) eliminated by capsaicin, indicating that it was solely of primary sensory origin. We conclude that the primary sensory fibers innervating the IMG are differentially distributed to arterial and venous neurons and that the pattern of distribution is characteristic for each sensory neurotransmitter.

Innervation of the rat pineal gland by pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibres.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibres were demonstrated in the rat pineal gland. These fibres entered the pineal gland through the conarian nerve at the distal tip of the gland. A high density of the fibres was observed in the capsule of the gland, from where the immunoreactive elements penetrated into the pineal perivascular spaces and parenchyma. The majority of PACAP-immunoreactive nerve fibres also contained calcitonin gene-related peptide (CGRP). Some PACAP-immunoreactive nerve fibres contained neuropeptide Y (NPY), but only occasionally was PACAP colocalized with vasoactive intestinal peptide (VIP). After removal of both superior cervical ganglia, a high number of PACAP-containing nerve fibres were still present in the gland. In the nervous system PACAP is present in two isoforms, PACAP-38 and PACAP-27. The concentration of PACAP-38 in the superficial pineal gland was determined by radioimmunoassay to be 20.4 pmol/g tissue at midday and 18.9 pmol/g tissue at midnight. The concentration of PACAP-27 was only about 3% of the concentration of PACAP-38. In summary, this study is the first demonstration of a PACAP-containing innervation of the rat pineal gland. The PACAP concentration in the pineal gland does not exhibit a day-night difference. The colocalization of PACAP with calcitonin gene-related peptide in the pincalopetal nerve fibres indicates that the majority of PACAP-immunoreactive nerve fibres might originate from the trigeminal ganglion.

Stability and plasticity of primary afferent projections following nerve regeneration and central degeneration.

Sensory neurons of the dorsal root ganglia (DRG) regenerate their peripheral axons with relative ease following a nerve lesion. The capacity for central regeneration appears more limited. However, after nerve lesion, some DRG neurons gain a regenerative advantage to sprout centrally. We developed a lesion model in the rat to test whether, after prior lesion of their peripheral axons, subsets of cutaneous afferents benefit differently in their ability to sprout into adjacent spinal segments denervated by dorsal rhizotomy. We found that under identical circumstances, myelinated sensory neurons, small-diameter peptidergic sensory neurons containing calcitonin gene related peptide (CGRP), and small-diameter nonpeptidergic neurons that bind the lectin from the plant Griffonia simplificolia, isolectin B4 (IB4) differ dramatically in their ability to regenerate centrally. Myelinated afferent terminals labelled transganglionically with cholera-toxin beta-subunit gain a small advantage in collaterally sprouting into the adjacent denervated neuropil in lamina III after prior peripheral nerve lesion. This central regenerative response was not mimicked by experimentally induced inflammation of sensory neuron cell bodies. Intact and unlesioned sensory neurons positive for CGRP sprout vigorously into segments denervated by rhizotomy in a nonsomatotopic manner. In contrast, IB4-positive sensory neurons maintain a somatotopic distribution centrally, which is not altered by prior nerve lesion. These data reveal a remarkably heterogeneous response to regeneration-promoting stimuli amongst three different types of cutaneous sensory neurons. In particular, the divergent responses of peptidergic and nonpeptidergic sensory neurons suggests profound functional differences between these neurochemically distinct neurons.

Expression of calcitonin gene-related peptide-1 receptor mRNA in human tooth pulp and trigeminal ganglion

Numerous nerve fibres containing calcitonin gene-related peptide (CGRP) were found by immunocytochemistry in human molar pulp. These nerves were often seen around small blood vessels and as free endings without vascular contact. In the trigeminal ganglion a large number of CGRP-immunoreactive nerve-cell bodies, mostly of small to medium size, was encountered. Reverse transcriptase-polymerase chain reaction, using specific sense and antisense primers, detected mRNA expression of the human CGRP 1 receptor in the pulp tissue and the trigeminal ganglion. Thus, both CGRP-containing nerve fibres and CGRP 1 receptor mRNA are present in human tooth pulp, where they may be involved in the regulation of vascular tone and other local reactions to injury.

A new concept of skin-associated lymphoid tissue (SALT): UVB light impaired cutaneous immunity reveals a prominent role for cutaneous nerves.

More than 20 years have passed since the concept that the skin has its own associated immune system was first proposed by Streilein. This proposal was advanced in part on evidence that cutaneous contact hypersensitivity (CH) reactions are closely correlated with Langerhans cells (LC). Recent reports have demonstrated that LC have neural connectivity with cutaneous nerve termini containing calcitonin gene-related peptide (CGRP), suggesting that a link exists between innervation and immune responses in the skin. Here we discuss the neural components which have recently been found to be participants in skin-associated lymphoid tissue (SALT). In part, discovery of a functional link between the nervous system and SALT is based on studies in which cutaneous immunity was impaired by ultraviolet-B radiation (UVR). The deleterious effects of UVR on cutaneous immunity include failed CH induction and promotion of hapten-specific tolerance, effects that are mediated by tumor necrosis factor-alpha and interleukin-10, respectively. The source of these cytokines after UVR appears to be dermal mast cells. Evidence indicates that mast cells are triggered to release these cytokines in response to CGRP, which is released from UVR-damaged cutaneous nerve endings. Moreover, a substance P agonist was able to reverse the deleterious effects of UVR on CH induction, rendering the mice able to develop intense CH. These observations indicate that two cell types not originally included in the SALT concept are critical to the functional integrity of cutaneous immunity: mast cells and cutaneous nerves. We propose that cutaneous nerves dictate whether antigen applied to or arising within skin will lead to sensitivity or tolerance.

Androgen receptor immunoreactivity is present in primary sensory neurons of male rats.

We have examined the distribution of androgen receptor (AR) immunoreactivity in L6 and S1 dorsal root ganglia of male rats in order to determine whether the sensory component of reflex circuits is likely to be androgen-sensitive. Nuclear AR immunoreactivity was present in almost half of the neurons, but was decreased markedly by castration; after castration nuclear staining was absent and a few neurons showed dim cytoplasmic staining. Of the neurons possessing AR, half also contained calcitonin gene-related peptide (CGRP); in turn, > 80% of CGRP neurons contained AR. AR staining was present in both large and small CGRP neurons. This study shows that testosterone is likely to influence many sensory neurons and may therefore play an important role in modulating visceral and somatic reflexes.

Temperature- and capsaicin-sensitive nerve fibers in brown adipose tissue attenuate thermogenesis in the rat.

We examined the function of putative sensory fibers that are contained in intercostal nerves and innervate interscapular brown adipose tissue (IBAT) in urethane-anesthetized rats. Warming the IBAT to 40-44 degrees C with two small heaters placed bilaterally on the skin above it attenuated the subsequent noradrenaline-induced thermogenesis (NIT) of the IBAT. In this range of warming, higher IBAT temperatures resulted in more attenuation. Denervation of IBAT blocked the effect of thermal stimulation on the NIT. Thus, activation of nerve fibers in IBAT that are sensitive to warmth or to the nociceptive effects of heat probably attenuated the NIT. Since the putative sensory fibers in the IBAT contain calcitonin gene-related peptide (CGRP) and substance P, which are thought to act in peripheral tissues, we tested the effects of injection of these neuropeptides into the IBAT. Administration of 5.2 nmol CGRP but not substance P or vehicle saline mimicked the effect of thermal stimulation of IBAT. As the neuropeptide-containing primary sensory neurons are characterized by their sensitivity to capsaicin, we also tested its effects (1 mg/kg, s.c.) and found that it also attenuated the NIT. Denervation of the IBAT or pretreatment with capsazepine, a capsaicin receptor antagonist, blocked the effect of capsaicin. We propose that temperature- and capsaicin-sensitive nerve fibers release CGRP to attenuate the NIT of brown adipocytes.

Presence of putative neurotransmitters in the myenteric plexus of the gastrointestinal tract and in the musculature of the urinary bladder of the ferret.

The innervation of the musculature in the ferret stomach, ileum, colon and urinary bladder was investigated using immunohistochemistry in noncolchicin-treated tissues. In the gastrointestinal tract two main subpopulations of myenteric neurones were found: cholinergic neurones expressing choline acetyltransferase (ChAT), which made up 68, 67 and 67% of the neurones in the stomach, ileum and colon, respectively, and nitrergic neurones containing nitric oxide synthase and NADPH-diaphorase (stomach: 23%, ileum: 21%, colon: 26%). In the stomach, cholinergic neurones expressed substance P (SP, 2% of all neurones), dopamine-beta-hydroxylase (DBH, 19%) but not tyrosine hydroxylase (TH) or vasoactive intestinal polypeptide (VIP), while nitrergic neurones contained VIP and neuropeptide Y (NPY). TH- but not DBH-immunoreactivity was observed in 4% of gastric neurones. Intense immunoreactivity in the musculature suggests that part of ChAT/SP- and NOS/NPY/VIP-positive neurones function as motorneurones. In the ileum, a high number (32%) of DBH-positive neurones was demonstrated. About half of the SP-positive neurones in the ileum also contained calcitonin gene-related peptide (CGRP). In the urinary bladder, only few intramural ganglia were observed. The smooth muscle was densely innervated by ChAT, NPY and DBH immunoreactive fibres. The data showed that the innervation of the ferret viscera exhibited similarities but also differences as compared with other mammalian species. Some of the chemical coding of myenteric neurones is remarkably similar to that observed in other mammals.

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)--in different functional conditions. IBAT was obtained from adult rats (6 weeks old) acclimated at different temperatures (4 degrees, 20 degrees, and 28 degrees 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.

Calcitonin gene-related peptide is necessary for ultraviolet B-impaired induction of contact hypersensitivity.

Since nerve termini on Langerhans cells (LC) contain calcitonin gene-related peptide (CGRP), and since ultraviolet B radiation (UVR) causes CGRP to be released from cutaneous nerve endings, we examined whether CGRP participates in the immune aberrations caused in skin by UVR. First, intradermally injected CGRP, in a dose-dependent manner, reduced LC density and impaired CH induction when hapten was painted on the injected site. Second, CGRP antagonist restored CH induction after UVR. Third, anti-TNF-alpha Abs injected before CGRP prevented the loss of LC density and restored CH induction. Fourth, CGRP failed to impair CH induction in mast cell-deficient mice. Fifth, CGRP induced mast cells to release TNF-alpha. We conclude that CGRP plays an essential role in the loss of CH induction after UVR. These data indicate that UVR, by causing the release of CGRP from cutaneous nerve endings, triggers mast cell release of TNF-alpha, which impairs CH induction.

Co-localization of nitric oxide synthase, neuropeptides and tyrosine hydroxylase in nerves supplying the human post-natal vas deferens and seminal vesicle.

To determine the distribution and patterns of co-localization of nitric oxide synthase (NOS), neuropeptides and tyrosine hydroxylase (TH) in intrinsic nerves of the human post-natal vas deferens and seminal vesicle. Double and triple immunolabelling methods were used in tissue from 10 male infants and children (age range 2 months to 3 years) obtained at post-mortem examinations carried out within 12 h of death. Most nerves supplying the muscle coat of either organ were TH-immunoreactive (-IR), most of which also contained neuropeptide Y (NPY) while a smaller proportion contained both NPY and NOS. Minor populations of the TH/NPY-IR intramuscular nerves contained calcitonin gene-related peptide (CGRP), galanin (GAL), met-enkephalin (m-ENK) or vasoactive intestinal polypeptide (VIP). Non-TH-IR intramuscular nerves were relatively infrequent and most contained NPY and either VIP or NOS. Presumptive secretomotor nerves formed subepithelial plexuses in both organs, most of which contained NPY co-localized with either VIP or NOS, with minor populations containing CGRP and/or GAL. TH- and substance P (SP) -IR nerves were not observed subepithelially. Perivascular nerve plexuses were mainly formed by TH-IR varicose nerves, most of which contained co-localized NPY and CGRP, with a smaller proportion containing NPY and NOS and minor populations containing VIP, m-ENK, SP or GAL. These results indicate that the autonomic control of the human vas deferens and seminal vesicle is provided by several immunohistochemically distinct nerve populations. Furthermore, NOS is present in a proportion of both the noradrenergic and non-noradrenergic nerves. Pharmacological studies are now required to elucidate the precise roles of nitric oxide and neuropeptides in the functional control of these organs.

Effects of several denervation procedures on distribution of calcitonin gene-related peptide and substance P immunoreactive in rat stomach.

The effect of chemical deafferentation, vagotomy (VGX), and gangliosympathectomy (GSX) on the density of fibers containing calcitonin gene-related peptide (CGRP) and substance P (Sub.P) in the rat gastric wall was studied. Chemical deafferentation by capsaicin abolished the density of CGRP-immunoreactive (IR) fibers, not Sub.P-IR fibers. Ten days after VGX, the density of CGRP-IR or Sub.P-IR fibers in the mucosa was largely reduced, while no reduction of CGRP-IR and Sub.P-IR fibers was seen in submucosal and muscular layers. GSX significantly reduced the density of CGRP-IR fibers in the mucosa and caused a moderate decrease in the fibers in submucosal and muscular layers. Pretreatment with 6-hydroxydopamine, a neurotoxin for noradrenergic nerves, did not affect the density of CGRP-IR fibers in the gastric wall. The density of Sub.P-IR fibers in the gastric wall was not affected by GSX. These studies indicate that the CGRP-IR and Sub.P-IR fibers in the mucosa are susceptible to extrinsic nerve denervation compared with those in the submucosa and muscle layers, that a major portion of the CGRP-IR fibers in the mucosa is of both vagal and spinal origin, and that a major portion of the Sub.P-IR fibers in the mucosa is of vagal origin. Furthermore, the present results support that CGRP-IR fibers, not Sub.P-IR fibers, in the rat stomach are capsaicin-sensitive.

Selective depletion of clear synaptic vesicles and enhanced quantal transmitter release at frog motor nerve endings produced by trachynilysin, a protein toxin isolated from stonefish (Synanceia trachynis) venom.

Our previous observation that low concentrations of stonefish (Synanceia trachynis) venom elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals prompted our present study to purify the quantal transmitter-releasing toxin present in the venom and to characterize the toxin's ability to alter the ultrastructure and immunoreactivity of frog motor nerve terminals. Fractionation of S. trachynis venom by sequential anion exchange fast protein-liquid chromatography (FPLC) and size-exclusion FPLC yielded a highly purified preparation of a membrane-perturbing (haemolytic) protein toxin, named trachynilysin. Trachynilysin (2-20 micrograms/ml) significantly increased spontaneous quantal acetylcholine release from motor endings, as detected by recording miniature endplate potentials from isolated frog cutaneous pectoris neuromuscular preparations. Ultrastructural analysis of nerve terminals in which quantal acetylcholine release was stimulated to exhaustion by 3 h exposure to trachynilysin revealed swelling of nerve terminals and marked depletion of small clear synaptic vesicles. However, trachynilysin did not induce a parallel depletion of large dense-core vesicles. Large dense core vesicles contained calcitonin gene-related peptide (CGRP), as revealed by colloidal gold immunostaining, and trachynilysin-treated nerve endings exhibited CGRP-like immunofluorescence similar to that of untreated terminals. Our results indicate that the ability of stonefish venom to elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals is a function of trachynilysin, which selectively stimulates the release of small clear synaptic vesicles and impairs the recycling of small clear synaptic vesicles but does not affect the release of large dense-core vesicles. Trachynilysin may be a valuable tool for use in other secretory terminals to discriminate between neurotransmitter and neuropeptide release.

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: an immunocytochemical study

Changes in the expression of several neurochemical markers associated with either axonal growth (GAP-43), synaptic vesicles (synaptophysin), or the cholinergic population of lateral olivocochlear (OC) efferents were investigated in the postnatal cochlea of hamsters. Growth-associated protein was expressed in the neonatal cochlea but not in the adult; immunoreactivity was found below inner hair cells (IHCs) from postnatal day (P) 2 through P14 and below outer hair cells (OHCs) from P5 to P14. In contrast, synaptophysin was expressed in both the neonate and adult cochlea; immunoreactivity was found below IHCs around P4 and below OHCs at P5. Both GAP-43 and synaptophysin immunoreactivities occurred first below IHCs in basal regions of the cochlea. Efferent fibers containing calcitonin gene-related peptide (CGRP) immunoreactivity were identified as early as P4 within the cochlear nerve but were not observed underneath IHCs until P7. Similar to GAP-43 and synaptophysin immunoreactivity, CGRP expression followed a basal to apical gradient; however, expression below OHCs appeared restricted to apical regions. These data raise the possibility that efferents expressing growth proteins and efferents expressing synaptic vesicle proteins co-exist during the first postnatal week. Furthermore, it is hypothesized that CGRP-containing lateral OC neurons from part of a later, secondary innervation to the cochlea.

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: an immunocytochemical study

Changes in the expression of several neurochemical markers associated with either axonal growth (GAP-43), synaptic vesicles (synaptophysin), or the cholinergic population of lateral olivocochlear (OC) efferents were investigated in the postnatal cochlea of hamsters. Growth-associated protein was expressed in the neonatal cochlea but not in the adult; immunoreactivity was found below inner hair cells (IHCs) from postnatal day (P) 2 through P14 and below outer hair cells (OHCs) from P5 to P14. In contrast, synaptophysin was expressed in both the neonate and adult cochlea; immunoreactivity was found below IHCs around P4 and below OHCs at P5. Both GAP-43 and synaptophysin immunoreactivities occurred first below IHCs in basal regions of the cochlea. Efferent fibers containing calcitonin gene-related peptide (CGRP) immunoreactivity were identified as early as P4 within the cochlear nerve but were not observed underneath IHCs until P7. Similar to GAP-43 and synaptophysin immunoreactivity, CGRP expression followed a basal to apical gradient; however, expression below OHCs appeared restricted to apical regions. These data raise the possibility that efferents expressing growth proteins and efferents expressing synaptic vesicle proteins co-exist during the first postnatal week. Furthermore, it is hypothesized that CGRP-containing lateral OC neurons from part of a later, secondary innervation to the cochlea.

Peptide-containing nerve fibres in normal human parathyroid glands and in human parathyroid adenomas

There are only a few studies on the innervation of the human parathyroid glands and the content of neurotransmitters. We therefore studied the occurrence and distribution of peptide-containing and adrenergic nerve fibres and the coexistence pattern of neuromessengers by immunocytochemistry in normal (unaffected) and adenomatous parathyroid glands from patients undergoing surgery for parathyroid adenoma. The unaffected parathyroid glands had a moderate-to-rich supply of nerve fibres and terminals containing two general neuronal markers, protein gene product 9.5 (PGP 9.5) and synaptophysin, neuropeptide Y (NPY) and tyrosine hydroxylase (TH). They were seen close to blood vessels and, occasionally, among the endocrine cells. Only a few nerves contained calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), substance P (SP) and pituitary adenylate cyclase-activating peptide (PACAP). The general density of innervation, using PGP 9.5 and synaptophysin as markers, varied greatly among the different adenomas examined. This applied also to the density of fibres and terminals containing specific types of messengers. Some of the tumours had a rich supply of TH- and NPY-containing nerve fibres, while others contained only few scattered fibres. The CGRP-containing fibres varied from moderate in number to no detectable fibres. The PACAP-, SP- and VIP-containing fibres were always very few or not detectable. It is not inconceivable that the wide variation in general density of the innervation and frequency of peptide-containing nerves among individual parathyroid adenomas is of significance for their hormone secretory behaviour.