Histochemical observations on cholinesterase activity in the autonomic ganglia of the human sympathetic trunk and vagus nerve

Synopsis The distribution of cholinesterase activity was studied histochemically in the autonomic ganglia of the human sympathetic trunk and the vagus nerve using a modified Koelle's technique. It was found that the cytoplasm of both sympathetic and parasympathetic nerve cells contained acetylcholinesterase but the intensity of the enzyme reaction varied from cell to cell in both types of ganglia. Tissue elements surrounding the nerve cells showed a low butyrylcholinesterase activity in the ganglia of the sympathetic trunk but a high one in the terminal ganglia of the vagus nerve. Postganglionic nerves fibres gave a weak reaction for acetylcholinesterase in the sympathetic, but a strong one in the vagus ganglia. The distribution pattern of cholinesterases in human autonomic ganglia was found to be different from that of a variety of laboratory and wild animals.     

E-cadherin expression in a particular subset of sensory neurons.

We found that the dorsal root ganglia (DRG) and trigeminal ganglia of mouse embryos express the E-cadherin cell-cell adhesion molecule and analyzed its expression profile. E-cadherin expression began around Embryonic Day 12 (E12) in these ganglia, thereafter increased, and persisted to the adult stage. This cadherin was expressed by 10 and 30% of DRG neurons in E17 and postnatal animals, respectively, as well as by satellite cells and some Schwann cells. E-cadherin-positive primary sensory fibers terminated only in a narrow region of the dorsal horn of the spinal cord, which was identified as part of lamina II by double-staining for E-cadherin and substance P or somatostatin. This E-cadherin expressing area of the spinal cord extended to part of the trigeminal nucleus in the medulla. These results showed that E-cadherin is expressed in a particular subset of primary sensory neurons which may have specific functional properties. We suggest that this adhesion molecule may play a role in the selective adhesion of sensory neuronal fibers.     

Non-specific cholinesterase activity in mouse spinal ganglia. The usefulness of histochemical study and image analysis for simple characterization of neuron subclasses

Non-specific cholinesterase (ChE) activity was studied histochemically at light and electron microscopical levels in dorsal root ganglia (DRG) of adult mice. The reaction staining and diameter of neuron cells perykaria were measured by using an image analysis system. The methodological approach enable to distinguish 8 subclasses of primary sensory neurons. The proportion of individual subclasses was mapping in three subsequent cervical, thoracal and lumbar DRG. The populations of small-sized neurons increased towards lumbar level similarly as medium and small neurons exhibiting high ChE reactivity. The variations in ChE-containing neurons among DRG from different area may reflect differences in modality-specific primary sensory neurons at each spinal cord level. In addition, the effect of 3 week sciatic nerve transection on the percentage of the subclasses in L4-L6 DRG has been investigated. The number of large neurons was reduced and a decrease of ChE reactivity in medium-size neurons was found in DRG on the operated side. Thus, the present results demonstrate a selective affectation of primary sensory neurons in mouse DRG by the peripheral nerve transection. Different amounts of the reaction product corresponding with ChE activity were found in the nuclear envelope and the cisternae of rough endoplasmic reticulum.     

Growth hormone-releasing factor (GRF)-like immunoreactivity in sensory ganglia of the rat

Summary Growth hormone-releasing factor (GRF)-like immunoreactivity has been demonstrated in the trigeminal and spinal ganglia of fetal, young and adult rats by use of peroxidase-antiperoxidase immunohistochemistry. GRF-like-immunoreactive cells first appear during the second half of embryonic life, as early as day 17. In untreated animals the GRF-immunoreactive elements form approximately 1% of all ganglion cells in the trigeminal and spinal ganglia; their numbers do not change significantly during development. The granular immunoreaction product is confined to perikarya, especially to the perinuclear region. Nerve fibers displaying GRF-like immunoreactivity were found neither in the ganglia, nor in the corresponding central and peripheral areas of termination. The possible role of GRF in sensory ganglia is discussed.     

The development of histochemically demonstrable cholinesterases in the rat neostriatum in vivo and in vitro

Summary The development of acetylcholinesterase (AChE) and non-specific cholinesterase (NsChE) activity was studied in the rat neostriatum by the light and electron microscope using three thiocholine methods. The AChE activity was first demonstrable only in the lateral parts of the nucleus, and during the early postnatal development the most intense activity was in the cell bodies, whilst the typical intense staining of the neuropil of adult animals was seen in two-week-old rats. Two types of AChE-containing cells were observed in the neostriatum of rats younger than two weeks and in cultures of newborn rat neostriatal cells. The neuropil of the cultures showed weak activity in the membranes of thin preterminal processes. In the neuropil of old rats, NsChE activity was present in the membranes of nerve cell processes. The capillary endothelial cells of newborn rats contained both AChE and NsChE. During subsequent development, the AChE activity disappeared, whilst for NsChE no change was seen in the distribution of activity seen in newborn or young adult rats less than three months old.     

The development of histochemically demonstrable cholinesterases in the rat neostriatum in vivo and in vitro

The development of acetylcholinesterase (AChE) and non-specific cholinesterase (NsChE) activity was studied in the rat neostriatum by the light and electron microscope using three thiocholine methods. The AChE activity was first demonstrable only in the lateral parts of the nucleus, and during the early postnatal development the most intense activity was in the cell bodies, whilst the typical intense staining of the neuropil of adult animals was seen in two-week-old rats. Two types of AChE-containing cells were observed in the neostriatum of rats younger than two weeks and in cultures of newborn rat neostriatal cells. The neuropil of the cultures showed weak activity in the membranes of thin preterminal processes. In the neuropil of old rats, NsChE activity was present in the membranes of nerve cell processes. The capillary endothelial cells of newborn rats contained both AChE and NsChE. During subsequent development, the AChE activity disappeared, whilst for NsChE no change was seen in the distribution of activity seen in newborn or young adult rats less than three months old.     

Tyrosinase Protein Is Expressed Also in Some Neural Crest Derived Cells Which Are Not Melanocytes

Some neural crest cells give rise to pigment cells in early ontogenesis. We tested here whether tyrosinase-a key enzyme in melanogenesis—was present in some nonpigment neural crest derivatives in adult hamsters. Interestingly enough, inactive tyrosinase protein was detected, using indirect immunofluorescence, in the satellite cells of spinal ganglia and Schwann cells of sciatic and facial nerves in normal adult animals. The results of cell blotting from spinal ganglia were similar to the fluorescence findings. Thus, our results seem to support the hypothesis that Schwann cells, satellite cells of spinal ganglia, and melanocytes may be more intimately related developmentally than other neural-crest-derived cells. Moreover, since we detected tyrosinase protein in cells which normally do not produce melanosomes, it could be deduced that, during the melanocyte's differentiation from its cell precursor, the expression of tyrosinase protein might precede the point when melanosomes begin to differentiate from known cytoplasmic structures.     

Inducible Prrxl1-CreER(T2) recombination activity in the somatosensory afferent pathway

Prrxl1-CreER(T2) transgenic mice expressing tamoxifen-inducible Cre recombinase were generated by modifying a Prrxl1-containing BAC clone. Cre recombination activity was examined in Prrxl1-CreER(T2); Rosa26 reporter mice at various embryonic and postnatal stages. Pregnant mice were treated with a single dose of tamoxifen at embryonic day (E) 9.5 or E12.5, and X-gal staining was performed 2 days later. Strong X-gal staining was observed in the somatosensory ganglia (e.g., dorsal root and trigeminal ganglia) and the first central sites for processing somatosensory information (e.g., spinal dorsal horn and trigeminal nerve-associated nuclei). When tamoxifen was administered at postnatal day (P) 20 or in adulthood (P120), strong Cre recombination activity was present in the primary somatosensory ganglia, while weak Cre recombination activity was found in the spinal dorsal horn, mesencephalic trigeminal nucleus, principal sensory trigeminal nucleus, and spinal trigeminal nucleus. This mouse line provides a useful tool for exploring genes' functions in the somatosensory system in a time-controlled way.     

Down-regulating effect on cellular immunity of the NSF17 factor produced by murine neonatal spleen cells fused with BW 5147 thymoma cells.

The non-specific factor NSF17 produced by mouse newborn splenocytes fused with BW 5147 thymoma cells was tested in vivo for its immunosuppressive activity. NSF17 administered i.p. in a single dose into adult mice significantly decreased the PHA-induced proliferative response of lymphocytes, regional graft-versus-host reaction, and delayed-type hypersensitivity.     

Ontogenic development of the reactivity of macrophages to antigenic stimulation

Experiments were performed to test the ability of macrophages from newborn mice to participate in immune reactions. It was found that peritoneal cells from 4-day-old mice injected at birth with thioglycollate did not reconstitute reactivity to Shigella in adult, irradiated mice, while normal adult macrophages did.The total yield of peritoneal exudate cells (PEC) was relatively low, yet the number of precursor cells of macrophages in the newborn spleen was found in significantly higher concentrations than in the adult. The number of precursor cells in the spleens of 0–3-day-old mice did not increase in response to antigenic stimulation, indicating that they too are unable at this stage to develop reactivity to immunological signals.     

Interferons produced by the cells of newborn mice in the presence of sera from newborn and adult animals

Fibroblasts of newborn mice produced far less amount of interferon in the presence of sera from newborn animals than in the presence of sera from adult animals. The interferons obtained were purified by adsorption chromatography on porous glass and were analyzed by electrophoresis in polyacrylamide gel. It has been shown that antiviral activity of interferon preparations obtained in the presence of sera from newborn mice was associated with the fraction of 45 Kd. Addition into the growth medium of sera from adult animals led to the production by the same cells of interferon activity associated with 41 and 28 Kd fractions. It is assumed that the sera of newborn mice contained the components influencing the molecular content of interferon produced by the cells of newborn animals.     

Spleen cells from adult mice given total lymphoid irradiation (TLI) or from newborn mice have similar regulatory effects in the mixed leukocyte reaction (MLR). II. Generation of antigen-specific suppressor cells in the MLR after the addition of spleen cells from newborn mice

Spleen cells from newborn BALB/c mice were added to the mixed leukocyte reaction (MLR) between a variety of responder and stimulator cells. The newborn cells nonspecifically suppressed the uptake of (3H)-thymidine and the generation of cytolytic cells regardless of the responder-stimulator combination used. Suppressor cell activity fell rapidly during the first 4 days after birth, and could not be detected by day 20. Newborn spleen cells inhibited the generation of nonspecific suppressor cells during the MLR but did not inhibit the generation of antigen-specific suppressor cells. Thus, newborn spleen cells exhibit a pattern of regulation of the MLR similar to that reported previously for spleen cells from adult mice given total lymphoid irradiation (TLI). These regulatory interactions provide a model that explains the ease of induction of transplantation tolerance in vivo in newborn mice and in TLI-treated adult mice.     

Binding Patterns of Lectins with GalNAc specificity in the mouse dorsal root ganglia and spinal cord

To localize membrane glycoconjugates in neurons of the mouse spinal cord and dorsal root ganglia (DRG), cryostat sections of newborn (P0), 7 day-old (P7), P14, P21 and P31 animals were stained with ten FITC-conjugated plant lectins, the majority of them recognizing N-acetyl-D-galactosamine (GalNAc) terminal sugar residues. In the dorsal root ganglia of P0 animals, the different lectins showed distinct patterns of labeling in either cells of the nervous system, including neurons, or other structures such as nerves or blood vessels. Moreover, some of these lectins showed important changes in their pattern of labeling during postnatal development. This was especially relevant for lectins that label a subpopulation of small-sized cells that have been previously identified as the nociceptive cells of the DRG. Enzymatic digestion of sections with neuraminidase removes sialic acid from the carbohydrate chains of glycoconjugates thus exposing novel sugar residues. When this treatment was applied to DRG sections from postnatal animals the pattern of lectin staining was either changed or eliminated and heterogeneous subsets of glycoconjugates normally masked by this sugar were exposed. In the spinal cord of PO animals, none of the lectins labeled cells in the central gray matter. However, after the enzymatic digestion of sections with neuraminidase, spinal cord motoneurons and some other cells were labeled by two of the lectins suggesting that GalNAc residues present in these cells are normally masked by terminal sialic acid. Altogether, these results show important changes in the temporal and spatial expression of glycoconjugates that may be relevant for the postnatal development of the CNS and PNS of mice.     

Cholinergic innervation of the cornea and iris of the guinea pig

Cholinergic innervation of the cornea and iris of the newborn and adult guinea pig was studied by the technique of Karnovsky and Roots (1964). The given structures are both richly innervated. The cholinesterase reaction of the cornea is more strongly positive in adult animals, whereas the intensity of the reaction of the iris in newborn and adult guinea pigs is almost identical.     

DEVELOPMENTAL CHANGES OF CHOLINESTERASES AND MONOAMINE OXIDASE IN CHICK EMBRYO SPINAL AND SYMPATHETIC GANGLIA

Abstract— Total cholinesterase, acetylcholinesterase, (AChE) and monoamine oxidase (MAO) activity and protein content were determined throughout the embryonic life of the chick in spinal and sympathetic ganglia. The greatest part of total cholinesterase activity was due to AChE.
AChE and MAO activity increased in both spinal and sympathetic ganglia very similarly from the 6th to the 12th day of incubation; from this day on a significant divergence occurred, mainly owing to a steady fall in spinal ganglion AChE, which decreased to approximately one tenth of the maximum value. The ratio of MAO activity in sympathetic and spinal ganglia increased from the 8th day onwards and approached 5·0 at hatching. The ratio between sympathetic and spinal ganglia, for AChE, choline acetylase (ChAc) and MAO activity, suggests a relationship between the maturation of the synapse in the sympathetic ganglia and the maximal activity of these enzymes.     

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.     

Morphological innervation pattern of the developing rabbit heart.

The morphological innervation pattern of developing fetal and neonatal rabbit hearts was delineated histochemically by a cholinesterase/silver procedure and immunohistochemically with the monoclonal antibody HNK1, an antibody which recognizes some cells derived from neuroectoderm. Cholinesterase-containing nerves appeared distally on the outflow tract by gestational day 15 (G15). Isolated cells with cholinesterase-stained fine processes were present near the base of the pulmonary trunk. HNK1 antibody stained the same nerves and ganglia revealed by the cholinesterase reaction and other nerves in the rabbit heart. It was used to confirm that cells with fine neuron-like processes were present before nerve ingrowth. The G14 heart contained many HNK1 staining cells in the right atrium, outflow, and inflow tracts; cells with fine processes were few but increased at G16. By G17, a plexus of interweaving nerves and associated cells began to form at the base of the pulmonary trunk. Fine nerves encircled the base of the aorta, and others crossed the intercaval region dorsally. At G19, nerves 1) extended downward from a rich "bulbar" plexus along the front ventricular surface, 2) grew near the epicardial surface at the base of the heart along the atrial floor and ventricular roof, 3) traversed the vena cavae and intercaval region to enter the atrial roof, and 4) crossed the coronary sinus to reach the back ventricular walls. By G23, cholinesterase-staining nerves and ganglia in the atria and, epicardially, in the ventricles formed the general innervation pattern of the newborn and adult rabbit heart.     

Cholinesterases and postnatal development of the negative chronotropic effects of acetylcholine in albino rats

The negative chronotropic effects of acetylcholine were studied in the isolated atria of the hearts of albino rats aged 2, 15, 29 and 47 days and adult. In untreated preparations, i.e. with full cholinesterase activity, the strongest effects were observed in newborn animals; with advancing age the reaction grew weaker. If cholinesterase activity was inhibited with physostigmine, the differences between the various age groups were obliterated. It is thus evident that the actual acetylcholine sensitivity of the sinoatrial node tissue does alter during postnatal life, but that growing cholinesterase activity reduces the amount of acetylcholine diffusing from the medium into the acetylcholine receptor zone. The change which takes place in cholinesterase activity in the myocardial tissue during postnatal life is so great that is must be taken into account when considering the development of cholinergic control of cardiac function.     

Target-derived BMP signaling limits sensory neuron number and the extent of peripheral innervation in vivo

The role of target-derived BMP signaling in development of sensory ganglia and the sensory innervation of the skin was examined in transgenic animals that overexpress either the BMP inhibitor noggin or BMP4 under the control of a keratin 14 (K14) promoter. Overexpression of noggin resulted in a significant increase in the number of neurons in the trigeminal and dorsal root ganglia. Conversely, overexpression of BMP4 resulted in a significant decrease in the number of dorsal root ganglion neurons. There was no significant change in proliferation of trigeminal ganglion neurons in the noggin transgenic animals, and neuron numbers did not undergo the normal developmental decrease between E12.5 and the adult, suggesting that programmed cell death was decreased in these animals. The increase in neuron numbers in the K14-noggin animals was followed by an extraordinary increase in the density of innervation in the skin and a marked change in the pattern of innervation by different types of fibers. Conversely, the density of innervation of the skin was decreased in the BMP4 overexpressing animals. Further Merkel cells and their innervation were increased in the K14-noggin mice and decreased in the K14-BMP4 mice. The changes in neuron numbers and the density of innervation were not accompanied by a change in the levels of neurotrophins in the skin. These findings indicate that the normal developmental decrease in neuron numbers in sensory ganglia depends upon BMP signaling, and that BMPs may limit both the final neuron number in sensory ganglia as well as the extent of innervation of targets. Coupled with prior observations, this suggests that BMP signaling may regulate the acquisition of dependence of neurons on neurotrophins for survival, as well as their dependence on target-derived neurotrophins for determining the density of innervation of the target.     

Comparison of [3H]resiniferatoxin binding to spinal cord and dorsal root ganglia of newborn and adult rats

Capsaicin is frequently used in neurobiological investigations to selectively inhibit response by the primary sensory afferent neurons. The effectiveness of treatment depends significantly on the age of the animals; newborns are both quantitatively and qualitatively more sensitive than adults. In the present study, we used the [³H]resiniferatoxin binding assay to determine whether this different susceptibility to capsaicin between newborns and adult animals may reflect differences either in receptor affinity or density. We report here that whole spinal cord membranes of neonates bound [³H]RTX with similar affinity and positive cooperativity as did the spinal cord membranes from adult animals (K_d values were 24.8 ± 3.7 and 26.8 ± 4.8 pM, respectively; Hill coefficients were 2.25 ± 0.03 and 2.17 ± 0.05, respectively). However, the receptor density was three - fold higher in the spinal cord membranes of neonates than of adult rats (B_max values were 142 ± 13 and 43 ± 3 fmol/mg protein, respectively). We found no significant difference in the [³H]RTX binding properties of dorsal root ganglia membranes of newborn and adult animals. Our results suggest than a higher density of the vanilloid receptor in the spinal cord (but not in the dorsal root ganglia) of newborn animals may contribute to the quantitative differences between the sensitivity of adult animals and neonates.