Effects of spinal cord transection on synapse numbers and biochemical maturation in rat lumbar sympathetic ganglia

The effects of interruption of descending central pathways on the morphological and biochemical ontogeny of peripheral sympathetic ganglia were examined in the sixth lumbar (L6) sympathetic rat ganglia. Previous studies defined the normal maturation of presynaptic choline acetyltransferase (CAT) activity, postsynaptic tyrosine hydroxylase (T-OH) activity, and total protein in the L6 ganglion. In the present investigations ganglion synapse numbers and adrenergic neuron numbers were examined in transected and sham-operated littermate controls 6 weeks after surgery at 10 days of age and correlative biochemical studies were performed. Spinal transection resulted in a reduction in synapse number on ganglion cell bodies (53.5% reduction) and neuronal processes (55.8% reduction). This abnormality in synapse number was associated with a reduction of CAT activity to 56% of control. Although T-OH activity also failed to develop normally and was 25% of control, there was no associated alteration in adrenergic neuron number. These studies suggest that descending central pathways regulate the maturation of presynaptic cholinergic terminals (and hence synapse number) in sympathetic ganglion and that CAT activity serves as an accurate marker for these terminals. Additionally, the ontogeny of ganglion T-OH activity is altered by central lesions and this effect is not secondary to a decrease in adrenergic neuron number.     

The effects of nerve growth factor (NGF) and antiserum to NGF on the development of embryonic sympathetic neurons in vivo

The role of nerve growth factor (NGF) in the development of embryonic sympathetic neurons was examined in vivo. Individual mouse embryos received transuterine injections of NGF or antiserum to NGF (anti-NGF), and the effects on the superior cervical ganglion (SCG) were studied. Treatment with NGF at any gestational stage, from the time of ganglion aggregation to birth, increased ganglion tyrosine hydroxylase (T-OH) activity. Both the number of catecholaminergic neurons and T-OH activity per neutron were increased. Choline acetyltransferase (ChAc) activity was increased by NGF at early gestational stages, but not at later stages. These observations suggest that perikarya containing ChAc are responsive to NGF, whereas preganglionic nerve terminals are not. Treatment with anti-NGF rapidly and permanently decreased ganglion T-OH activity. The effects of anti-NGF were more pronounced at later gestational stages, suggesting that ganglia become increasingly dependent on NGF during development. Alteration of maternal levels of NGF had no effect on development of the embryonic SCG, suggesting that local embryonic concentrations of NGF are responsible for modulating sympathetic ontogeny.     

Environmental co-regulation of substance P, somatostatin and neurotransmitter synthesizing enzymes in cultured sympathetic neurons

Mechanisms regulating peptide neurotransmitter metabolism were examined in dissociated cell cultures of the neonatal rat superior cervical ganglion (SCG). The pineal gland, a target of the SCG, produced a soluble factor (PCM) which increased substance P (SP) levels more than 15-fold in sympathetic neurons cultured in the presence of ganglion non-neuronal cells. Elimination of the non-neuronal cells decreased SP to negligible levels and abolished the stimulatory effects of PCM on SP expression. These observations suggest that ganglion non-neuronal cells stimulate sympathetic expression of SP, and that the pineal influences neuronal SP by acting on, or in concert with, ganglion support cells. PCM also influenced other neurotransmitter systems. In the presence of ganglion non-neuronal cells, PCM treatment increased cholineacetyltransferase (CHAC) and decreased tyrosine hydroxylase (TOH) and somatostatin (SO). By contrast, PCM treatment of pure neuronal cultures resulted in negligibleCHAC and SP levels and a doubling of SO with a small increase in TOH. In sympathetic neurons, SP expression may be associated with cholinergic development, whereas SO may be associated with noradrenergic phenotypic expression. Moreover, there is a reciprocal relationship between SP and SS expression by sympathetic neurons analogous toe previously described relationship between noradrenergic and cholinergic expression^17–19.     

Prolonged sympathetic innervation of sensory neurons in rat thoracolumbar dorsal root ganglia during chronic colitis

Background Peripheral irritation‐induced sensory plasticity may involve catecholaminergic innervation of sensory neurons in the dorsal root ganglia (DRG). Methods Catecholaminergic fiber outgrowth in the thoracolumbar DRG (T13‐L2) was examined by tyrosine hydroxylase (TH) immunostaining, or by sucrose‐potassium phosphate‐glyoxylic acid histofluorescence method. TH level was examined by Western blot. Colonic afferent neurons were labeled by retrograde neuronal tracing. Colitis was induced by intracolonic instillation of tri‐nitrobenzene sulfonic acid (TNBS). Key Results The catecholaminergic fibers formed ‘basket‐like’ structures around the DRG cells. At 7 days following TNBS treatment, the number of DRG neurons surrounded by TH‐immunoreactive fibers and the protein levels of TH were significantly increased in T13, L1, and L2 DRGs (two‐ to threefold, P < 0.05). The DRG neurons that were surrounded by TH immunoreactivity were 200 kDa neurofilament‐positive, but not isolectin IB4‐positve or calcitonin gene‐related peptide‐positive. The TH‐immunoreactive fibers did not surround but adjoin the specifically labeled colonic afferent neurons, and was co‐localized with glial marker S‐100. Comparison of the level of TH and the severity of colonic inflammation showed that following TNBS treatment, the degree of colonic inflammation was most severe at day 3, subsided at day 7, and significantly recovered by day 21. However, the levels of TH in T13‐L2 DRGs were increased at both 3 days and 7 days post TNBS treatment and persisted up to 21 days (two‐ to fivefold increase, P < 0.05) as examined. Conclusions & Inferences Colonic inflammation induced prolonged catecholaminergic innervation of sensory neurons, which may have relevance to colitis‐induced chronic visceral hypersensitivity and/or referred pain.     

Postnatal development of neurons containing choline acetyltransferase in rat spinal cord: an immunocytochemical study

A monoclonal antibody to choline acetyltransferase (ChAT) has been used in an immunocytochemical study of the postnatal development of ChAT-containing neurons in cervical and thoracic spinal cord. Specimens from rat pups ranging in age from 1 to 28 days postnatal (dpn) were studied and compared with adult specimens (Barber et al., '84). The development of established cholinergic neurons, the somatic motoneurons and sympathetic preganglionic cells, has been described as has that of previously unidentified ChAT-positive neurons in the dorsal, intermediate, and central gray matter. Cell bodies of somatic and visceral motoneurons contained moderate amounts of ChAT-positive reaction product at birth that gradually increased in intensity until 14-21 dpn. The most intensely stained ChAT-positive neurons in 1-5-dpn specimens were named partition cells because this cell group extended from the central gray to an area dorsal to the lateral motoneurons, and thereby divided the spinal cord into dorsal and ventral halves. Partition cells were medium to large in size with 5-7 primary dendrites, and axons that, in fortuitous sections, could be traced into the ventrolateral motoneuron pools, the ventral funiculi, or the ventral commissure. Small ChAT-positive cells clustered around the central canal and scattered in laminae III-VI of the dorsal horn were detectable at birth. These neurons were moderately immunoreactive at 11-14 dpn and intensely ChAT positive by 21 dpn. The band of ChAT-positive terminal-like structures demonstrated in lamina III of adult specimens (Barber et al., '84) was first visible in 11-14-dpn specimens. By 28 dpn, both laminae I and III contained punctate bands that approximated the density of those observed in adult spinal cord. This investigation has demonstrated ChAT within individual neurons of developing spinal cord, and has identified a group of neurons, the partition cells, that exhibit intense ChAT-positive immunoreactivity earlier than any other putative cholinergic cells in spinal cord, including motoneurons. Another important observation has been that each ChAT-positive neuronal type achieves adult levels of staining intensity at different times during development. A likely explanation for this differential staining is that various groups of neurons acquire their mature concentration of ChAT molecules at different developmental stages. In turn, this may correlate with the maturation of cholinergic synaptic activity manifest by individual cells or groups of neurons.     

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 subpopulation 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. © 1994 Wiley-Liss, Inc.     

Topography of choline acetyltransferase Immunoreactive Neurons and fibers in the rat spinal cord

The topography of choline acetyltransferase immunoreactivity was studied in the rat spinal cord with a monoclonal antibody. Cholinergic fibers were most prominent in lamina III of the dorsal horn and originated from cholinergic neurons within the spinal cord. Lamina X, which was rich in cholinergic neurons and fibers, provided cholinergic interconnections between the dorsal, intermediate and ventral gray. Within the ventral gray, choline acetyltransferase immunoreactive boutons were found on motor neurons. This study suggests that the cholinergic innervation of the spinal cord arises from neurons intrinsic to the spinal cord. The cholinergic neurons within the spinal cord may provide several, overlapping levels of regulation of spinal cord neurons.     

Topographical localization of choline acetyltransferase within the human spinal cord and a comparison with some other species

Choline acetyltransferase (ChAT), which is known to be a specific marker of cholinergic structures, was assayed in small tissue samples punched out from cryosections of human, bovine, cat and rat spinal cords. The relative distribution patterns of spinal ChAT were similar between the different species. An area of high activity in the ventrolateral part of the ventral horn was found. This activity is probably located in the motor neurons, as it could be traced into the ventral root region. In addition, in the dorsal horn of the cord from man and cow another area with high ChAT activity was found. Subcellular studies suggest that this activity is mainly located at nerve terminals.     

Trans-synaptic increase in RNA coding for tyrosine hydroxylase in a rat sympathetic ganglion

To begin examining molecular mechanisms underlying trans-synaptic regulation, tyrosine hydroxylase (TH) and its messenger RNA (mRNA) were examined in the superior cervical sympathetic ganglion (SCG) of adult rats. Basal levels of TH mRNA were detectable in control ganglia by RNA dot hybridization, using the ³²P nick translated PstI-KpnI restriction fragment of pTH.4 as a probe. Reserpine induced a 3-fold rise in TH activity per μg protein, and a simultaneous 3-fold increase in ganglion TH mRNA. As expected, ganglion decentralization (denervation) prevented the trans-synaptic induction of TH. In addition, decentralization prevented the increase in TH mRNA, suggesting that the increase in message was dependent on trans-synaptic stimulation. Northern blot analysis indicated that the cDNA (complementary DNA) probe hybridized to a single band of approximately 1900 nucleotides, which was the same size in all ganglia. Our observations indicate that induction of TH is associated with a trans-synaptic increase in mRNA coding for the enzyme. Consequently, trans-synaptic increases in impulse activity may induce TH by increasing neuronal levels of TH mRNA in the SCG.     

Characterization of oxytocin immunoreactivity in human sympathetic paravertebral ganglia

Immunoreactive oxytocin (IR-OXT) detected in extracts of human lumbar sympathetic paravertebral ganglia was characterized by high-performance liquid chromatography (HPLC). The immunoreactive substance was found to elute at the same position as the reference preparation of oxytocin (OXT). The results revealed the presence of chromatographically identified OXT in human sympathetic ganglia.     

Estimation of conduction velocity in bulbospinal excitatory pathways to sympathetic outflows in cat spinal cord

Experiments were performed on cats to measure the conduction velocity of the bulbospinal sympatho-excitatory axons. The lateral white matter of the cervical spinal cord and regions of the medulla were systematically explored for sympatho-excitatory points whilst recording from two sympathetic preganglionic outflows simultaneously. These were chosen a suitable distance apart to give a clear difference in latency to enable precise calculation of axonal conduction velocity between the two outflows. In addition, in a few cases conduction velocity was also measured by determining the latency shift of evoked responses, resulting from moving the stimulating electrode a known distance closer to the recording site. Conduction velocities of sympatho-excitatory axons in the spinal cord determined by these two procedures lay in the range 1.6-7.9 m/s.     

Anaplastic lymphoma kinase is dynamically expressed on subsets of motor neurons and in the peripheral nervous system

During embryonic development, complex events, such as cellular proliferation, differentiation, survival, and guidance of axons, are orchestrated and regulated by a variety of extracellular signals. Receptor tyrosine kinases mediate many of these events, with several playing critical roles in neuronal survival and axonal guidance. It is evident that not all the receptor tyrosine kinases that play key roles in regulating neuronal development have been identified. In this study, we have characterized the spatial-temporal expression profile of a recently identified receptor tyrosine kinase, anaplastic lymphoma kinase (ALK), in embryonic chick by means of whole-mount in situ hybridization in conjunction with immunohistochemistry. Our findings reveal that Alk is expressed in sympathetic and dorsal root ganglia as early as stage 19. In addition, mRNA is expressed from stage 23/24 (E4) to stage 39 (E13) in discrete motor neuron subsets of chick spinal cord along with a select group of muscles that are innervated by one of these particular motor neuron clusters. Expression within the spinal cord is coincident with the onset and duration of motor neuron programmed cell death and during the period of musculature innervation and synapse formation. Hence, the data presented here identify ALK as a novel candidate receptor for regulating critical events in the development of neurons in both the central and the peripheral nervous systems.     

Evidence for the coexistence of acetylcholine and enkephalin in the sympathetic preganglionic neurons of rats

The localization of the cholinergic neurons in the lower thoracic segments of the spinal cord of rats was examined by a monoclonal antibody against choline acetyltransferase (ChAT). The ChAT-immunoreactive neurons were located in the intermediate as well as anterior gray matters. In the intermediate gray the highest incidence of the immunoreactive neurons was in the nucleus intermediolateralis, followed by the nucleus intercalatus pars paraependymalis and a few immunoreactive neurons were seen in the nucleus intercalatus proprius. In the sequential immunostaining of one and the same section of the spinal cord pretreated with colchicine using the ChAT antibody and a polyclonal antibody against methionine-enkephalin-argynine-glycine-leucine (Met-Enk-Arg-Gly-Leu), substantial numbers of neurons were immunostained simultaneously by the two antibodies in the intermediate gray matter. The present finding gives strong evidence for the coexistence of acetylcholine and enkephalins in, at least, some of the preganglionic neurons projecting their axons to the periphery.     

Cholinergic and monoaminergic innervation of the cat's thalamus: comparison of the lateral geniculate nucleus with other principal sensory nuclei

The cholinergic and monoaminergic innervation of the lateral geniculate nucleus (GL) and other thalamic nuclei in the cat was examined by using immunocytochemical and tract-tracing techniques. Cholinergic fibers, identified with an antibody to choline acetyltransferase (ChAT), are present in all layers of the GL. They are fine in caliber and exhibit numerous swellings along their lengths. The A layers, the magnocellular C layer, and the medial interlaminar nucleus are rich in cholinergic fibers that give rise to prominent clusters of boutons, while the parvicellular C layers contain fewer fibers that are more uniformly distributed. The interlaminar zones are largely devoid of ChAT-immunoreactive fibers. Double-label experiments show that cholinergic projections to the GL originate from two sources, the pedunculopontine reticular formation (PPT) and the parabigeminal nucleus (Pbg). The PPT contributes cholinergic fibers to all layers, while Pbg projections are limited to the parvicellular C layers. The lateral geniculate nucleus has a much greater density of cholinergic fibers than the other principal sensory nuclei: the density of fibers in the A layers is more than three times greater than that in the ventral posterior nucleus (VP) or the ventral division of the medial geniculate nucleus (GMv). In contrast, serotonin (5-HT)-immunoreactive fibers are distributed with equal density across the principal thalamic nuclei, while tyrosine hydroxylase (TH)-immunoreactive fibers (presumed to contain norepinephrine) are noticeably less dense in the GL than in the others. Monoaminergic fibers also differ from cholinergic fibers in their laminar distribution within the GL: both TH- and 5HT-immunoreactive fibers are distributed evenly across the layers and interlaminar zones and are slightly more abundant in the parvicellular C layers than in the other layers. Other thalamic nuclei rich in cholinergic fibers include the pulvinar nucleus, the ventral lateral geniculate nucleus, the intermediate nucleus of the lateral group, the lateral medial and suprageniculate nuclei (Graybiel and Berson: Neuroscience 5:1175-1238, '80), and the paracentral and central-lateral components of the intralaminar nuclei. This pattern matches the distribution of projections from the PPT and is similar, but not identical, to the pattern of acetylcholinesterase staining. The fact that most of the nuclei rich in cholinergic fibers have been implicated in visual sensory or visual motor functions suggests that cholinergic projections from the reticular formation play an especially important role in visually guided behavior.     

Neurotransmitter synthesis by SN6 cell lines,a family of hybrid cell lines of embryonic septal origin

Previously, we reported the presence of multiple neurotransmitters in subclones of SN6, a septal cholinergic hybrid cell line. To obtain information concerning the functionality of these transmitters, we measured transmitter contents, activities of transmitter-producing enzymes, and the effect of serum-free culture medium in two different batches (SN6.1.6 and SN6.10.2.2) and two subclones of the SN6 cell line (SN6.2a and SN6.1b). Except for SN6.1b, SN6 cell lines and subclones had basically the same neurotransmitter characteristics. Among the transmitters, only acetylcholine seemed to be functional. Monoamine oxidase was missing and activity of aromatic amino acid decarboxylase was diminished in SN6 cell lines. Even in serum-containing medium, SN6.1b had a more mature morphology than the other cell lines, and it contained choline acetyltransferase and acetylcholine but not tyrosine hydroxylase or catecholamines. Similar characteristics were acquired by the mother cell line in response to serum-free conditions. Thus, SN6.1b is the most mature of these central cholinergic neuronal cell lines, at least with regard to neurotransmitter profiles. ©1995 Wiley-Liss, Inc.     

Selective modulation of cholinergic properties in cultures of avian embryonic sympathetic ganglia

We have studied the expression of catecholaminergic and cholinergic phenotypes in sympathetic ganglia removed from 7- to 10-day-old quail embryos and grown in vitro under different conditions. Quantitative data were obtained by measuring the conversion of (³H) tyrosine and (³H) choline to catecholamines (CA) and acetylcholine (ACh), respectively. In explant cultures, large amounts of both neurotransmitters were synthesized from the onset, but CA generally predominated, the molar ratios of CA:ACh being, on average, of the order of 2:1. If the ganglia were dissociated before plating, there was a selective increase in ACh synthesis (three- to fivefold) such that the CA:ACh ratio fell strikingly. The early expression of the cholinergic phenotype appears to be species-specific in that, under identical conditions, dissociated cell cultures of newborn mouse superior cervical ganglia were overwhelmingly catecholaminergic (CA:ACh ratio of approximately 40:1) and ACh synthesis was only just detectable. Addition of veratridine (1.5 μM) either to explant or to dissociated cell cultures of embryonic quail sympathetic ganglia barely altered CA-synthesizing ability; in contrast, ACh synthesis and accumulation were stimulated about threefold. This effect, which we found to correspond to a quantitatively similar increase in the activity of choline acetyltransferase (ChAT), was completely blocked by tetrodotoxin, indicating that it was due to Na⁺-dependent depolarization. A preferential stimulation of ACh production was also observed when the concentration of K⁺ was raised to 20 mM. Veratridine treatment of cultures of presumptive sympathoblasts, in the form of sclerotome-associated neural crest cells, had identical effects. Our results reveal the quantitative importance of ACh-related properties in avian sympathetic ganglia from the earliest stages of their development and suggest that depolarization may be one of the factors selectively enhancing expression of the cholinergic phenotype during ontogeny. In these respects, the neurochemical differentiation of sympathetic neurons unfolds according to dissimilar scenarios in birds and mammals. © 1993 Wiley-Liss, Inc.     

Phenotypic plasticity of avian embryonic sympathetic neurons grown in a chemically defined medium: direct evidence for noradrenergic and cholinergic properties in the same neurons.

Avian embryonic sympathetic ganglia possess both catecholaminergic and cholinergic features and can synthesize noradrenaline (NAd) and acetylcholine (ACh) simultaneously. In the present study we sought to determine (1) whether or not this coproduction of NAd and ACh corresponds to the existence of two non-overlapping populations, and (2) to what extent the levels of synthesis are influenced by non-neuronal ganglion cells. We have focused on the correlation between the immunocytochemically demonstrable presence of the noradrenergic and cholinergic enzymes tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), respectively, and the synthesis of the corresponding neurotransmitters in embryonic quail sympathetic neuronal and non-neuronal cells purified by fluorescence-activated cell sorting. We show that (1) freshly sorted neurons synthesize both NAd and ACh, whereas non-neuronal cells produce neither; (2) the overwhelming majority of the sympathetic neurons display TH immunoreactivity; (3) about half of the TH-positive neurons are recognized by an anti-ChAT antibody in an artificial medium that selectively enhances synthesis and/or accumulation of ACh; (4) the non-neuronal cells are important for survival of the neurons and potentiate their synthesis of ACh in this medium, and (5) finally, we present evidence that expression of TH in noradrenergic neurons and in small intensely fluorescent cells of sympathetic ganglia is differentially regulated.     

Nestin expression defines both glial and neuronal progenitors in postnatal sympathetic ganglia

Sympathetic ganglia are primarily composed of noradrenergic neurons and satellite glial cells. Although both cell types originate from neural crest cells, the identities of the progenitor populations at intermediate stages of the differentiation process remain to be established. Here we report on the identification in vivo of glial and neuronal progenitor cells in postnatal sympathetic ganglia, by using mouse superior cervical ganglia as a model system. There are significant levels of cellular proliferation in mouse superior cervical ganglia during the first 18 days after birth. A majority of the proliferating cells express both nestin and brain lipid-binding protein (BLBP). Bromodeoxyuridine (BrdU) fate-tracing experiments demonstrate that these nestin and BLBP double-positive cells represent a population of glial progenitors for sympathetic satellite cells. The glial differentiation process is characterized by a marked downregulation of nestin and upregulation of S100, with no significant changes in the levels of BLBP expression. We also identify a small number of proliferating cells that express nestin and tyrosine hydroxylase, a key enzyme of catecholamine biosynthesis that defines sympathetic noradrenergic neurons. Together, these results establish nestin as a common marker for sympathetic neuronal and glial progenitor cells and delineate the cellular basis for the generation and maturation of sympathetic satellite cells.