Bernard Droz

Innervation of Putative Rapidly Adapting Mechanoreceptors by Calbindin- and Calretinin-immunoreactive Primary Sensory Neurons in the Rat

Calbindin and calretinin are two homologous calcium‐binding proteins that are expressed by subpopulations of primary sensory neurons. In the present work, we have studied the distribution of the neurons expressing calbindin and calretinin in dorsal root ganglia of the rat and their peripheral projections. Calbindin and calretinin immunoreactivities were expressed by subpopulations of large‐ and small‐sized primary sensory neurons and colocalized in a majority of large‐sized ones. The axons emerging from calbindin‐ or calretinin‐immunoreactive neurons innervated muscle spindles, Pacini corpuscles and subepidermal lamellar corpuscles in the glabrous skin, formed palisades of lanceolate endings around hairs and vibrissae, and gave rise to intraepidermal nerve endings in the digital skin. Since most of these afferents are considered as rapidly adapting mechanoreceptors, it is concluded that calbindin‐ or calretinin‐expressing neurons innervate particular mechanoreceptors that display physiological characteristics of rapid adaptation to stimuli.

Myelin-associated glycoprotein immunoreactive material: an early neuronal marker of dorsal root ganglion cells during chick development

Immunostaining of myelin-associated glycoprotein (MAG) was performed in chick dorsal root ganglia (DRG) during development. The MAG-immunoreactive material appeared first around 7 days of incubation in immature neurons of DRG. Immunoprecipitates first confined to one pole of nucleus were gradually redistributed in the perinuclear Golgi apparatus of small DRG cells. Thus MAG may be used in the chick embryo as an early marker of primary sensory neurons of class B.

Carbonic anhydrase activity in primary sensory neurons

SummarySubpopulations of primary sensory neurons in mammalian dorsal root ganglion (DRG) exhibit carbonic anhydrase (CA) activity. To identify these subpopulations in DRG cells of mouse and chicken, the reliability of the cytochemical localization of the enzyme requires that several conditions be fulfilled:(1) Preservation of the enzyme activity in glutaraldehyde-containing fixative; (2) accessibility of the cytoenzymatic reaction throughout 20-μm thick Vibratome sections; (3) retention of the reaction product in situ during OsO4 post-fixation; (4) specificity of the cytoenzymatic reaction for CA activity as corroborated by the immunocytochemical detection with antibodies anti-CA II in mouse DRG; (5) strict correlation between the CA activity and the cytological characteristics in a given subclass of neurons. On the basis of these criteria, it is concluded that the CA activity may be used as a cell marker to identify cytologically defined neuronal subpopulations and their axons in mouse DRG. In chicken DRG, CA activity is not consistently expressed in a given subclass of ganglion cells and their axons. Hence, it is assumed that the expression of CA activity by DRG cells in chicken is modulated by functional or environmental conditions.

Substance P-like-immunoreactive sensory neurons in dorsal root ganglia of the chick embryo: ontogenesis and influence of peripheral targets

The expression of substance P (SP) was studied in sensory neurons of developing chick lumbosacral dorsal root ganglia (DRG) by using a mixture of periodic acid, lysine and paraformaldehyde as fixative and a monoclonal antibody for SP-like immunostaining. The first SP-like-immunoreactive DRG cells appeared first at E5, then rapidly increased in number to reach a peak (88% of ganglion cells) at E8, and finally declined (59% at E12, 51% after hatching). The fall of the SP-like-positive DRG cells resulted from two concomitant events affecting a subset of small B-neurons: a loss of neuronal SP-like immunoreactivity and cell death. After one hindlimb resection at an early (E6) or late (E12) stage of development (that is before or after establishment of peripheral connections), the DRG were examined 6 days later. In both cases, a drastic neuronal death occurred in the ispilateral DRG. However, the resection at E6 did not change the percentage of SP-like-positive neurons, while the resection at E12 severely reduced the proportion of SP-like-immunoreactive DRG cells (25%). In conclusion, connections established between DRG and peripheral target tissues not only promote the survival of sensory neurons, but also control the maintenance of SP-like-expression. Factors issued from innervated targets such as NGF would support the survival of SP-expressing DRG cells and enhance their SP content while other factors present in skeletal muscle or skin would hinder SP expression and therefore lower SP levels in a subset of primary sensory neurons.

Peripheral projections of calretinin-immunoreactive primary sensory neurons in chick hindlimbs.

In chicken dorsal root ganglia, calretinin immunoreactivity is expressed by a subpopulation of large A-neurons, most of which co-express calbindin D-28k. The myelinated axons of these neurons selectively innervate all muscle spindles and most Herbst corpuscles associated to feathers in hindlimbs. It is suggested that the presence of calretinin in primary afferents may be correlated with the electrophysiological properties of rapidly adapting mechanoreceptors.

The expression of nuclear 3,5,3′ triiodothyronine receptors is induced in Schwann cells by nerve transection

The effects of thyroid hormones on the nervous system are mediated by the presence of nuclear T3 receptors (NT3R). In this study, the expression of NT3R was investigated in spinal cord, dorsal root ganglia (DRG), or sciatic nerve of adult rats after immunostaining with a 2B3-NT3R monoclonal antibody which recognizes both alpha and beta types of NT3R. The specificity of this monoclonal antibody was confirmed by Western blots. The 2B3-NT3R monoclonal antibody recognized one band corresponding to a molecular weight of 57 kDa in extract of spinal cord or DRG. No staining was observed on immunoblot of intact sciatic nerve. In the spinal cord, the nuclei of the neurons and glial cells including both astrocytes and oligodendrocytes exhibited 2B3-NT3R immunoreactivity. While all the nuclei of the DRG sensory neurons expressed the NT3R, all the nuclei of the satellite and Schwann cells were devoid of any immunoreaction. In the sciatic nerve, the nuclei of the Schwann cells also lacked 2B3-NT3R-immunoreactivity. After sciatic nerve transection in vivo, Schwann cell nuclei, which never expressed NT3R in intact nerves of adult rats, displayed a clear 2B3-NT3R immunoreaction in proximal and distal stumps adjacent to the section. Double immunostaining with antibodies raised to 3-sulfogalactosylceramide or S100 confirmed that most of the NT3R containing nuclei belong to Schwann cells. In dissociated cell cultures grown in vitro from sciatic nerves, Schwann cells exhibited 2B3-NT3R immunoreactivity. These data suggest that the inhibition of NT3R expression in Schwann cells ensheathing axons in intact nerve is reversed when the axons are degenerating or lacking.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of Calbindin Immunoreactivity by Subpopulations of Primary Sensory Neurons in Chick Embryo Dorsal Root Ganglion Cells Grown in Coculture or Conditioned Medium

Primary sensory neurons which innervate neuromuscular spindles in the chicken are calbindin-immunoreactive. The influence exerted by developing skeletal muscle on the expression of calbindin immunoreactivity by subpopulations of dorsal root ganglion (DRG) cells in the chick embryo was tested in vitro in coculture with myoblasts, in conditioned medium (CM) prepared from myoblasts and in control cultures of DRG cells alone. Control cultures of DRG cells grown at the 6th embryonic day (E6) did not show any calbindin-immunostained ganglion cell. In coculture of myoblasts previously grown for 14 days, about 3% of calbindin-immunoreactive ganglion cells were detected while about 1% were observed in some cultures grown in CM. Fibroblasts from various sources were devoid of effect. Skin or kidney cells were more active than myoblasts to initiate calbindin expression by subpopulations of DRG cells in coculture or, to a lesser degree, in CM. The results suggest that cellular factors would rather induce calbindin expression in certain sensory neurons than ensure a selective neuronal survival.

Influence of environmental factors on the expression of phenotypic characters by chicken dorsal root ganglion cells in culture.

Primary sensory neurons were grown under four conditions of culture. The influence of nonneuronal cells, horse serum or both was studied on the phenotypic expression of certain neuronal subpopulations. The number of neurons expressing acetylcholinesterase, alpha-bungarotoxin-binding sites or a high uptake capacity for glutamine was enhanced by nonneuronal cells. The horse serum increases the neuronal subpopulation exhibiting a carbonic anhydrase activity. Certain phenotypic changes fit conditions consistent with an epigenetic induction rather than a cell selection.

Calbindin-immunoreactive sensory neurons in dissociated dorsal root ganglion cell cultures of chick embryo: role of culture conditions

Immunoreactivity to calbindin D-28k, a vitamin D-dependent calcium-binding protein, is expressed by neuronal subpopulations of dorsal root ganglia (DRG) in the chick embryo. To determine whether the expression of this phenotypic characteristic is maintained in vitro and controlled by environmental factors, dissociated DRG cell cultures were performed under various conditions. Subpopulations of DRG cells cultured at embryonic day 10 displayed calbindin-immunoreactive cell bodies and neurites in both neuron-enriched or mixed DRG cell cultures. The number of calbindin-immunoreactive ganglion cells increased up to 7-10 days of culture independently of the changes occurring in the whole neuronal population. The presence of non-neuronal cells, which promotes the maturation of the sensory neurons, tended to reduce the percentage of calbindin-immunoreactive cell bodies. Addition of horse serum enhanced both the number of calbindin-positive neurons and the intensity of the immunostaining, but does not prevent the decline of the subpopulation of calbindin-immunoreactive neurons during the second week of culture; on the contrary, the addition of muscular extract to cultures at 10 days maintained the number of calbindin-expressing neurons. While calbindin-immunoreactive cell bodies grown in culture were small- or medium-sized, no correlation was found between cell size and immunostaining density. At the ultrastructural level, the calbindin immunoreaction was distributed throughout the neuroplasm. These results indicate that the expression of calbindin by sensory neurons grown in vitro may be modulated by horse serum-contained factors or interaction with non-neuronal cells. As distinct from horse serum, muscular extract is able to maintain the expression of calbindin by a subpopulation of DRG cells.

Preferential Synthesis of Prostaglandin D2 by Neurons and Prostaglandin E2 by Fibroblasts and Nonneuronal Cells in Chick Dorsal Root Ganglia

Abstract: To determine the type and the relative amount of prostaglandins (PGs) synthesized by various neural tissues, homogenates of meninges, dorsal root ganglia (DRG) capsules, decapsulated DRG, and unsheathed sciatic nerves were incubated with [1‐14C]arachidonic acid. Homogenates of cultured cells (meningeal cells, fibroblasts, and nonneuronal or neuronal DRG cells) were used to specify the cells producing particular PGs. The highest synthetic capacity was found in fibroblast‐rich tissues (meninges and DRG capsules) and in cultures of meningeal cells or fibroblasts. Two major cyclooxygenase products were formed: [14C]PGE2 and an unusual 14C‐labeled compound, Y. The accumulation of compound Y, corresponding probably to 15‐hydroperoxy PGE2, was completely impaired by addition of exogenous GSH, which conversely enhanced the synthesis of [14C]PGE2and promoted the formation of [14C]PGD2. In contrast, decapsulated DRG or unsheathed sciatic nerves displayed a 10–20 times lower capacity to synthesize PGs than fibroblast‐rich tissues and produced mainly [14C]PGE2 and [14C]PGD2. In this case, [14C]PGE2 or [14C]PGD2 synthesis was neither enhanced nor promoted by addition of exogenous GSH. Neuron‐enriched DRG cell cultures allowed us to specify that [14C]PGD2 is the major prostanoid produced by primary sensory neurons as compared with nonneuronal DRG cells. Because PGD2 synthesis in DRG and more specifically in DRG neurons does not depend on exogenous GSH and differs from PGD2 synthesis in fibroblast‐rich tissues, it is concluded that at least two distinct enzymatic processes contribute to PGD2 formation in the nervous system. Although nonneuronal DRG cells give rise to both PGE2 and PGD2 and fibroblasts produce the largest amount of PGE2, it should be emphasized that PGD2 appears as the prostanoid preferentially, although not exclusively, synthesized by primary sensory neurons.