Muscarinic receptors modulate Nerve Growth Factor production in rat Schwann-like adipose-derived stem cells and in Schwann cells.

Regenerative capability of the peripheral nervous system after injury is enhanced by Schwann cells (SCs) producing several growth factors. The clinical use of SCs in nerve regeneration strategies is hindered by the necessity of removing a healthy nerve to obtain the therapeutic cells. Adipose-derived stem cells (ASCs) can be chemically differentiated towards a SC-like phenotype (dASCs), and represent a promising alternative to SCs. Their physiology can be further modulated pharmacologically by targeting receptors for neurotransmitters such as acetylcholine (ACh). In this study, we compare the ability of rat dASCs and native SCs to produce NGF in vitro. We also evaluate the ability of muscarinic receptors, in particular the M2 subtype, to modulate NGF production and maturation from the precursor (proNGF) to the mature (mNGF) form. For the first time, we demonstrate that dASCs produce higher basal levels of proNGF and mature NGF compared to SCs. Moreover, muscarinic receptor activation, and in particular M2 subtype stimulation, modulates NGF production and maturation in both SCs and dASCs. Indeed, both cell types express both proNGF A and B isoforms, as well as mNGF. After M2 receptor stimulation, proNGF-B (25 kDa), which is involved in apoptotic processes, is strongly reduced at transcript and protein level. Thus, we demonstrate that dASCs possess a stronger neurotrophic potential compared to SCs. ACh, via M2 muscarinic receptors, contributes to the modulation and maturation of NGF, improving the regenerative properties of dASCs.

Microvesicles and exosomes in metabolic diseases and inflammation.

Metabolic diseases are based on a dysregulated crosstalk between various cells such as adipocytes, hepatocytes and immune cells. Generally, hormones and metabolites mediate this crosstalk that becomes alterated in metabolic syndrome including obesity and diabetes. Recently, Extracellular Vesicles (EVs) are emerging as a novel way of cell-to-cell communication and represent an attractive strategy to transfer fundamental informations between the cells through the transport of proteins and nucleic acids. EVs, released in the extracellular space, circulate via the various body fluids and modulate the cellular responses following their interaction with the near and far target cells. Clinical and experimental data support their role as biomarkers and bioeffectors in several diseases includimg also the metabolic syndrome. Despite numerous studies on the role of macrophages in the development of metabolic diseases, to date, there are little informations about the influence of metabolic stress on the EVs produced by macrophages and about the role of the released vesicles in the organism. Here, we review current understanding about the role of EVs in metabolic diseases, mainly in inflammation status burst. This knowledge may play a relevant role in health monitoring, medical diagnosis and personalized medicine.

Influence of spermatozoal lipidomic profile on the cryoresistance of frozen spermatozoa from stallions.

The membrane of spermatozoa, which contributes to cellular cryoresistance, contains numerous lipids with a composition that directly affects membrane fluidity and the fertilization process. In light of variations in the degree of sensitivity in equine seminal freezing, this study aimed to correlate equine semen lipids with post-thawing characteristics of spermatozoa. We used ejaculates from 34 stallions, which were evaluated (total motility ≥ 60%), frozen and thawed and reevaluated for motility of spermatozoa, membrane integrity and lipid peroxidation. Lipid extraction of the fresh semen samples was performed by liquid-liquid extraction, and fingerprinting lipid analysis was conducted by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Based on the characteristics of spermatozoa after thawing, the animals could be separated into two groups: resistant (Good Freezers, n = 5) and sensitive (Bad Freezers, n = 6) to freezing, and their MALDI-MS data were then compared. The Good Freezers group showed a higher abundance of phosphatidylcholines (m/z 796.6, 846.6, 810.6, 854.6 and 732.6). The ions of m/z 812.6, 832.6, 836.6 and 838.6 belonging to the phosphatidylcholine lipid class were also positively correlated with motility of spermatozoa, whereas that of m/z 794.6 was negatively correlated with lipid peroxidation in thawed semen. The Bad Freezer group, displayed higher abundance of one phosphatidylcholines (m/z 806.6), as well as a sphingomyelins (m/z 703.5), which were negatively correlated (univariate analysis) with kinetics of spermatozoa after thawing (m/z 703.5) and with membrane integrity (m/z 792.6). The ion of m/z 717.5, assigned to phosphatidic acid, was negatively correlated with lipid peroxidation. In general therefore, the phosphatidylcholines are associated with higher quality of spermatozoa after thawing, especially in functional capacity, and that lipid semen composition was found to influence the resistance of spermatozoa to cryopreservation and may interfere with motility, membrane integrity and lipid peroxidation in stallions.

Lipid profiles of follicular fluid from cows submitted to ovarian superstimulation.

Ovarian superstimulation with exogenous gonadotropins has been extensively used to produce in vivo-derived embryos for embryo transfer in cattle. This process modifies the antral follicle microenvironment and affects oocyte and embryo quality as well the differentiation of granulosa cells. Lipids play significant roles in the cell, such as energy storage, cell structure, and fine-tuning of the physical properties and functions of biological membranes. The phospholipid (PL) contents as well as the effects of superstimulatory treatments on the PL profile of follicular fluid from cows, however, remain unknown. Therefore, to gain insight into the effects of superstimulation with follicle-stimulating hormone (FSH; P-36 protocol) or FSH combined with equine chorionic gonadotropin (eCG; P-36/eCG protocol) on the profile and abundance of PL from cows submitted or not submitted to superstimulatory protocols, were treated with these two superstimulatory protocols. As a control, non-superstimulated cows were only submitted to estrous synchronization. The follicular fluid was aspirated, the remaining cells removed and the follicular fluid stored at -80 °C until extraction. The lipid screening was performed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and this technique allowed the identification of sphingomyelins (SM) and phosphatidylcholines (PC) and phosphoethanolamines (PE). The relative abundance of the ions observed in the three experimental groups was analyzed by multivariate and univariate statistical models. The phospholipid SM (16:0) and PC (36:4) and/or PC (34:1) were less (P < 0.05) abundant in the P-36 group compared to the control or P-36/eCG groups. However, the PC (34:2) was more (P < 0.05) abundant in both group of superstimulated cows compared to the control. In summary, ovarian superstimulation seems to modulate the PL content of bovine follicular fluid with a significant increase in PC (34:2), which jointly with others PC and SM, seems to offer a suitable biomarker involved with reproductive processes successful as ovary superstimulation response and embryo development.

The mechanisms and possible sites of acetylcholine release during chick primary sensory neuron differentiation.

AIMS: In this study, we evaluated the ability of differentiating embryonic chick DRG neurons to release and respond to acetylcholine (ACh). In particular, we investigated the neuronal soma and neurites as sites of ACh release, as well as the mechanism(s) underlying this release. MAIN METHODS: ACh release from DRG explants in the Campenot chambers was measured by a chemiluminescent assay. Real-time PCR analysis was used to evaluate the expression of ChAT, VAChT, mediatophore and muscarinic receptor subtypes in DRGs at different developmental stages. KEY FINDINGS: We found that ACh is released both within the central and lateral compartments of the Campenot chambers, indicating that ACh might be released from both the neuronal soma and fibers. Moreover, we observed that the expression of the ChAT and mediatophore increases during sensory neuron differentiation and during the post-hatching period, whereas VAChT expression decreases throughout development. Lastly, the kinetics of the m2 and m3 transcripts appeared to change differentially compared to the m4 transcript during the same developmental period. SIGNIFICANCE: The data obtained demonstrate that the DRG sensory neurons are able to release ACh and to respond to ACh stimulation. ACh is released both by the soma and neurite compartments. The contribution of the mediatophore to ACh release appears to be more significant than that of VAChT, suggesting that the non-vesicular release of ACh might represent the preferential mechanism of ACh release in DRG neurons and possibly in non-cholinergic systems.

M2 muscarinic receptors inhibit cell proliferation in human glioblastoma cell lines.

AIMS: In the present work we investigated the expression of M2 muscarinic receptor subtype in two glioblastoma cell lines and its role in the control of cell proliferation. MAIN METHODS: The M2 receptor transcript and protein expression was studied using RT-PCR and western blot analysis. (3)[H]-thymidine incorporation was used to evaluate cell proliferation in the presence or in the absence of M(2) agonist arecaidine. KEY FINDINGS: We demonstrated that M(2) receptor is expressed in both cell lines, although U251 cells show a higher expression level, compared to U87 cells. The activation of M(2) receptors by the agonist arecaidine decreases cell growth in a dose and time dependent manner. The anti-proliferative effect of arecaidine is also confirmed by the significant decrease of (3)[H]-thymidine incorporation in both cell lines. Moreover the M2 antagonist gallamine counteracts the arecaidine effects confirming M2 receptor involvement in glioma cell growth inhibition. SIGNIFICANCE: These data suggest a role for M2 receptors in the inhibition of glioma cell proliferation and the possibility of exploiting these receptors as new promising tools for glioblastoma therapy.

Chemical composition of lipids present in cat and dog oocyte by matrix-assisted desorption ionization mass spectrometry (MALDI- MS).

The aim of the present study was to investigate the level of information on the chemical structures and relative abundances of lipids present in cat and dog oocytes by matrix-assisted laser desorption mass spectrometry (MALDI-MS). The MALDI-MS approach requires a simple analysis workflow (no lipid extraction) and few samples (two or three oocytes per analysis in this work) providing concomitant profiles of both intact phospholipids such as sphingomyelins (SM) and phosphatidylcholines (PC) as well as triacylglycerols (TAG). The lipids were detected in oocytes by MALDI using dihydroxybenzoic acid (DHB) as the matrix. The most abundant lipid present in the MS profiles of bitch and queen oocytes was a PC containing 34 carbons and one unsaturation [PC (34:1)]. Oocytes of these two species are characterized by differences in PC and TAG profiles detected qualitatively as well as by means of principal component analysis (PCA). Cat oocytes were mainly discriminated by more intense C52 and C54 TAG species and a higher number of unsaturations, indicating predominantly linoleic and oleic fatty acyl residues. Comparison of the lipid profile of bitch and queen oocytes with that of bovine oocytes revealed some similarities and also some species specificity: TAG species present in bovine oocytes were also present in bitches and queens; however, a more pronounced contribution of palmitic, stearic and oleic fatty acid residues was noticed in the lipid profile of bovine oocytes. MALDI-MS provides novel information on chemical lipid composition in canine and feline oocytes, offering a suitable tool to concomitantly monitor, in a nearly direct and simple fashion the composition of phospholipids and TAG. This detailed information is highly needed to the development of improved protocols for in vitro culture and cryopreservation of cat and dog oocytes.

Histamine activates a background, arachidonic acid-sensitive K channel in embryonic chick dorsal root ganglion neurons.

Histamine has been proposed to be an important modulator of developing neurons, but its mechanism of action remains unclear. In embryonic chick dorsal root ganglion neurons we found that histamine activates, through the pyrilamine-sensitive H1 receptor, a K-selective, background channel. The K channel activated by histamine was also activated by arachidonic acid in a dose-dependent way, with a KD of 4 microM and a slope of 2.5, had a unitary conductance of about 150 pS (symmetrical 140 KCl) and a moderate voltage dependence. The channel was insensitive to the classical K channel blockers tetraethylammonium, charybdotoxin, 4-aminopyridine, but inhibited by millimolar Ba2+. Channel activity could also be increased by lowering the intracellular pH from 7.2 to 5.5, or by applying negative pressure pulses through the patch pipette. Experiments aimed at delineating the metabotropic pathway leading to K channel activation by histamine indicated the involvement of a pertussis toxin-insensitive G protein, and a quinacrine-sensitive cytosolic phospholipase A2. The histamine-induced K channel activation was observed only with elevated internal Ca2+ (achieved using 0.5 microM ionomycin or elevated external KCl). An increase in the histamine-induced phosphoinositide hydrolysis was also observed upon internal Ca2+ elevation, showing the presence of a Ca2+ dependent step upstream to inositol 1,4,5-triphosphate production. In view of the functional importance of K conductances during cell differentiation, we propose that histamine activation of this K channel may have a significant role during normal development of embryonic chick neurons.

An in situ hybridization protocol to detect rare mRNA expressed in neural tissue using biotin-labelled oligonucleotide probes.

The use of the non-radioactive in situ hybridization protocols has allowed in general to obtain a better resolution of different transcripts at histological and cytological levels with a shortening of the developmental time. The common protocols using digoxigenin and biotin-labelled probes share a considerable limitation depending on the amount of the transcripts present in the tissues. This problem becomes more evident when oligonucleotide probes are used, because of their small size and lower ability to give sufficient signal amplification. The protocol reported here allows to localize rare mRNA expressed in a tissue, using a combination of two biotin-labelled oligonucleotide probes followed by streptavidin-peroxidase and biotinyl tyramide amplification system.

Acetylcholine synthesis and neuron differentiation.

Development of the nervous system is dependent on the co-operation between cell determination events and the action of epigenetic factors; in addition to well known factors, e.g. growth factors, neurotransmitters have been assigned a role as "morphogens" and modulators of neuronal differentiation in an early developmental phase. The possible role of acetylcholine as a modulator of neuronal differentiation has been considered in two experimental systems. A neuroblastoma cell line, which does not synthesise any neurotransmitter, has been transfected with a choline acetyltransferase construct; activation of acetylcholine synthesis, thus achieved, is followed by a higher expression of neuronal specific traits. The presence in these cells of muscarinic receptors is consistent with the existence of an autocrine loop, which may be responsible for the more advanced differentiation state observed in the transfected cells. Expression of cholinergic markers appears as a common feature of DRG sensory neurons, independently of the neurotransmitter used. Choline acetyltransferase can be detected in DRG at early developmental stages. The distribution of muscarinic receptors in DRG has suggested that activation of acetylcholine synthesis may be related in an early developmental phase to the interaction between neurons and nonneuronal cells and to modulation of cell differentiation. Both systems suggest that acetylcholine may have a role as a modulator of neuronal differentiation.

Modulation of cholinergic marker expression by nerve growth factor in dorsal root ganglia.

The presence of cholinergic markers in sensory ganglia has suggested a possible functional role of acetylcholine both as a cofactor of morphogenesis in embryonic life and in sensory transduction during adult life. Acetylcholine, in fact, is able to excite cutaneous nociceptors and to modulate noxious stimuli. Nerve growth factor (NGF) overexpression induces the survival of nociceptive neurons, the expression of their specific markers, and hyperalgesia. On the other hand, NGF modulate the levels of cholinergic markers in several area of nervous system. Considering these observations, the present work aims to investigate whether NGF is able also to control the expression of cholinergic markers in chick sensory neurons in culture. We selected three developmental stages (E8, E12, and E18) representative of different phases of chick embryo development and performed observations on culture in which NGF was omitted at the plating time, withdrawn after the initial 24 hr of culture or maintained for 48 hr. In the experimental protocol devised, NGF did not significantly affect cell survival. At E12 a 48 hr treatment with NGF causes a significant but limited increase in acetylcholinesterase activity; activity increase was not observed when NGF was removed after 24 hr. No changes in acetylcholinesterase activity were observed at E8 and E18 stages. NGF appears to be more effective in the modulation of choline acetyltransferase activity. At E12, in fact, about a doubling of enzyme activity was measured after 24 or 48 hr of treatment with NGF. A response was also found at E18, when a 50% increase in choline acetyltransferase activity was observed just after 24 hr treatment. The behavior of muscarinic receptors in response to NGF differs compared to the two cholinergic enzymes. At E8 and E12 a profound increase in muscarinic receptor expression was observed. Conversely, at E18 NGF produces a 50% reduction of receptors. Considering these observations and the demonstrated role of muscarinic receptors in the desensitization of nociceptors, the reduction of muscarinic receptors in DRG after NGF treatment is in agreement with the proposed algogenic action of NGF in the skin.

Muscarinic receptor subtypes expression in rat and chick dorsal root ganglia.

In the present work we have analyzed by Northern blot, RT-PCR and in situ hybridization the expression of muscarinic receptor subtype mRNAs in rat and chick dorsal root ganglia. Northern blot analysis performed on rat total RNA revealed a strong signal for M(2) while a faint band was observed for M(3) and M(4) subtypes; no signal was evident for M(1) and M(5), while in chick total RNA no signal was detected for any of the analyzed subtypes (M(2), M(3), M(4)). On the other hand, RT-PCR revealed that all muscarinic subtype mRNAs were present both in rat and chick DRG, although the level of their expression may be different. In chick DRG, the presence of various muscarinic subtypes was confirmed by competition binding experiments. In situ hybridization in rat DRG showed that M(3) and M(4) transcripts, similarly to what has been previously described for M(2) mRNA, were preferentially localized in medium-small neurons. Large neurons were usually negative or faintly labelled. No hybridization signal was detected in rat DRG with probes for M(1) and M(5) muscarinic subtypes. The presence of various muscarinic receptors in DRG and their preferential expression in the medium-small sensory neurons suggest their possible involvement in the modulation of nociceptive stimuli transduction.

Muscarinic receptors modulate intracellular calcium level in chick sensory neurons.

In the present work we have studied the variation of intracellular calcium levels induced by muscarinic agonists in chick dorsal root ganglia neurons. Muscarinic agonists such as muscarine and oxotremorine cause an increase of intracellular calcium levels in fura-2AM-loaded DRG neurons of E18 chick embryos. This increase was abolished following treatment with 1 microM atropine but not by 1 microM mecamylamine, indicating that the observed intracellular calcium increase, was dependent on muscarinic receptor activation. Stimulation in absence of external calcium or pre-incubation of the DRG cultures with thapsigargin or Mn(2+) demonstrated that [Ca(2+)](i) increase is mainly due to its release from intracellular stores. The use of selective antagonists of muscarinic receptor subtypes also indicated that M(1) and to a lesser extent M(3) receptor subtypes are responsible for the observed intracellular calcium mobilization. Finally pre-treatment of DRG cultures with pertussis toxin showed that the variation of [Ca(2+)](i) levels was dependent on PTX-insensitive G-protein. Moreover muscarinic agonists induce in DRG also the increase of IPs level, suggesting that the variations of intracellular calcium levels may be due at least in part to the activation of the phosphoinositide transduction pathway. In conclusion the reported observations demonstrate the activity of muscarinic receptors in sensory neurons, suggesting a functional role for acetylcholine in sensory transduction.

Expression of muscarinic m2 receptor mRNA in dorsal root ganglia of neonatal rat.

The expression of mRNA coding for m2 subtype of muscarinic cholinergic receptors was assessed in dorsal root ganglia (DRG) of 15-day post-natal rats. Northern blot analysis on total RNA using a mixture of two different oligonucleotide probes, indicated the presence of a single prominent band of approximately 6.5 kb in rat DRG; a band of the same size was observed both in brainstem and cortex taken as positive controls. Analysis by RT-PCR of the mRNA coding for a region of the third cytoplasmic loop of m2 receptor showed a single signal both in rat DRG and hippocampus. In situ hybridization was then used to identify the neuronal subpopulations expressing the mRNA for M2. The transcripts were preferentially localized in medium-small neurons of the ganglion as well as in satellite cells surrounding the neuron cell body. Large neurons were usually negative. Finally, competition binding experiments, performed in the presence of [3H]-quinuclidinyl benzilate (QNB) and methoctramine (a selective competitor for M2 receptors), demonstrated the presence of M2 receptor protein (Ki=100 nM), as previously observed in chick DRG. The preferential localization of M2 in medium-small neurons of the ganglion suggests the involvement of this receptor subtype in the transduction of nociceptive stimuli.

Neuronal and non-neuronal cell populations of the avian dorsal root ganglia express muscarinic acetylcholine receptors.

The distribution of muscarinic acetylcholine receptors was investigated by immuno-light and electron microscopy in the chick dorsal root ganglion during embryonic development (E12 and E18) and after hatching. The monoclonal antibody we used recognizes the acetylcholine binding site shared by all five muscarinic acetylcholine receptor subtypes. At E12, light microscopy reveals several immunopositive neurons with variable degrees of immunolabeling, heterogeneously distributed throughout the ganglion. Later in development and after hatching, the intensity of immunolabeling seems to decrease and the immunopositive neurons, of the small-medium-sized type, are located mostly in the medio-dorsal region of the ganglion. Under the electron microscope, the immunoreaction is associated with the Nissl bodies, budding Golgi cisterns and, especially at E12, with discrete loci along the neuronal plasma membrane. Unmyelinated nerve fibers, in both central and peripheral branches, are also immunopositive, suggesting that muscarinic acetylcholine receptors are transported towards the spinal cord and the periphery, respectively. A large number of perineuronal satellite cells and both myelinating and unmyelinating Schwann cells are intensely labeled. These observations, combined with previous data on the pharmacological and functional characterization of muscarinic acetylcholine receptors in the avian dorsal root ganglion, suggest that both sensory neurons and non-neuronal cells are able to respond to acetylcholine stimuli. Since muscarinic acetylcholine receptor-immunoreactivity is restricted to the small-medium-sized neurons and their unmyelinated fibers, of the nociceptive type, we suggest that these receptors are involved in modulating the transduction of noxious stimuli from the periphery.

Dystrophin localization and gene expression in the developing nervous system of the chick.

The presence and distribution of dystrophin was studied in selected areas of the chick embryo nervous system and in primary cultures. Dystrophin was examined at the protein level by immunocytochemistry and at the transcriptional level by a semiquantitative reverse transcriptase-polymerase chain reaction analysis. Immunofluorescence staining shows that dystrophin is present early during embryogenesis in dorsal root ganglia, spinal cord, and ciliary ganglia and colocalizes with neurofilament subunits. Cultured dorsal root ganglion, spinal cord, and ciliary ganglion neurons show immunoreactivity for dystrophin, both in cell bodies and along fibers. Dystrophin mRNA level in ciliary and dorsal root ganglia is higher than in spinal cord throughout development and shows a tissue-specific pattern of expression. In primary cultures of dorsal root ganglia and ciliary ganglia, dystrophin mRNA level increases with time in vitro. However, in spinal cord cultures, dystrophin mRNA drastically decreases with time in vitro, but it is significantly increased when embryonic muscle extract is added to the cultures. Our results show that dystrophin is present in neurons from different areas of embryonic chick nervous system and that its mRNA level is developmentally regulated both in vivo and in vitro.

Muscarinic cholinergic receptors in dorsal root ganglia of chick embryo: a radioligand binding and immunocytochemical study.

The presence and micro-anatomical localization of muscarinic cholinergic receptors were assessed in dorsal root ganglia of chick embryo during development using radioligand binding and immunocytochemical techniques, respectively. The non-selective muscarinic cholinergic receptor radioligand [3H]quinuclidinyl benzilate was specifically bound to sections of chick dorsal root ganglia with a dissociation constant value (Kd) of 0.75 +/- 0.02 nM and a maximum density of binding sites (Bmax) of 7.2 +/- 0.5 fmol/mg tissue. [3H]Quinuclidinyl benzilate binding was partially sensitive to pirenzepine displacement. This suggests that muscarinic cholinergic receptors expressed by dorsal root ganglia of chick embryo at least in part belong to the M1 muscarinic receptor subtype. Immunocytochemical analysis confirmed the presence of muscarinic receptors in the ganglia. These findings suggest that neurons of dorsal root ganglia, which are known to express cholinergic markers such as choline acetyltransferase, acetylcholinesterase and high affinity choline uptake, are also cholinoceptive.

Cholinergic markers are expressed in developing and mature neurons of chick dorsal root ganglia.

The presence of acetylcholinesterase has been reported in chick dorsal root ganglia at early developmental stages although acetylcholine is not known to play a role in these ganglia. Recently, we reported that during development the level of acetylcholinesterase increases continuously and the enzyme becomes gradually expressed in all sensory neurons. These observations prompted the study of the developmental pattern of expression of other cholinergic markers, such as choline acetyltransferase (ChAT) and the high affinity transport mechanism for choline. ChAT activity is barely detectable at early developmental stages (E7) and increases markedly thereafter, with an activity profile similar to that described for acetylcholinesterase. A similar increase in enzyme activity is also observed when ChAT is measured in dorsal root ganglia explants and in dissociated cells in culture. The study of ChAT activity in cultured cells shows an increase over a period of 3 days, thus ruling out the hypothesis that motor fibers, still associated to the ganglia, may represent a possible source of the enzyme. Immunostaining of whole ganglia or cultured cells shows that ChAT immunoreactivity is not restricted to a specific neuronal sub-population but appears as a common marker of sensory neurons. High affinity choline uptake, blocked by hemicholinium, is present in sensory neurons cultured from E7 dorsal root ganglia. Observations on cultured neurons from later stages (E18) indicate that choline transport is not a transient property of sensory neurons. These observations show a similar pattern of expression of several cholinergic markers during development. Such a pattern is maintained at significant levels also in mature ganglia.

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.