Acetylcholinesterase in the development of chick dorsal root ganglia

Acetylcholinesterase is expressed in chick dorsal root ganglia neurons very early in development. Since the physiological role of the enzyme in these cells is still obscure, it appeared of interest to investigate its modifications in the course of development. The specific activity of acetylcholinesterase in chick dorsal root ganglia increases, during in ovo development, from day E5 to day E13; after day E13 there is a decrease. Conversely, when acetylcholinesterase activity was expressed on a per ganglion basis, a continuous increase in the level of the enzyme until day E20 was observed. Acetylcholinesterase is a polymorphic enzyme and its molecular forms have different cellular localizations. Two globular forms, a tetramer (G4) and a dimer (G2), are present in the ganglia, as in chick brain. G4 is the major form at day E5, where it represents about 85% of the activity. This form shows a progressive decrease since day E8, and at day E20 exhibits activity levels similar to those of G2. It is known that acetylcholinesterase-producing cells are also able to release the enzyme in the extracellular space. We determined the release of acetylcholinesterase by cultured dorsal root ganglia neurons at various developmental stages: acetylcholinesterase release is significantly increased at day E20, as compared to younger stages, and 90% of the enzyme released is G4.     

Choline acetyltransferase and acetylcholinesterase in canine spinal ganglia: increase of choline acetyltransferase activity following sciatic nerve lesion

Activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were measured in the dorsal spinal ganglia, the dorsal spinal root and the spinal cord of the normal adult dogs and following one side transection of the sciatic nerve in the intervals 5, 10, 15 and 21 days respectively. In the spinal ganglia of normal dogs very low ChAT activity was found; it was three orders lower than AChE activity. Within 5-10 days after the nerve section ChAT activity increased almost five times in the spinal ganglia while AChE activity remained without any changes. The elevation of ChAT activity correlated with that in the dorsal roots at 15th day and in the dorsal spinal cord at 21st day after the nerve section. Histochemical "direct-colouring" thiocholine method showed AChE-positive cells were distributed mainly in the peripheral area of the spinal ganglia. The spinal ganglion cells ranged from intensely AChE-positive to AChE-negative without correlation between cell size and AChE activity. The ChAT activity changes were evaluated in correlation to the cholinergic function in the spinal ganglion neurons.     

Preferential cholinergic projections by embryonic spinal cord neurons within cocultured mouse superior cervical ganglia

The development of preferential cholinergic projections of spinal cord neurons within superior cervical ganglia (SCG) was analyzed in vitro using cocultures of SCGs (E17) with organotypic explants of fetal mouse cord (E13). The cord explants consisted of: (1) dorsal vs medioventral strips or mediodorsal vs ventral strips (dissected from levels C8-T4), or (2) transverse sections cut at various levels of the neuraxis. After 4 weeks of coculture, choline acetyltransferase (ChAT) was assayed in individual explants to quantify development of the cholinergic neurotransmitter enzyme (a) within the cord neurons, and (b) within the SCG. An index of cholinergic interaction was calculated as the relative ChAT activity in cocultured ganglion per unit ChAT activity in the ipsilateral cord strip. The highest index value (0.7) was obtained in cocultures with mediodorsal strips of cord. The index of interaction was progressively lower with medioventral (0.4), ventral (0.3) and dorsal (0.1) cord. In cocultures of transverse sections of spinal cord and SCGs, the highest indices of cholinergic interaction (expressed per hemisection of cord) were obtained with cord levels T1/T2 (1.0) and T5 (0.9). The index decreased with T9 (0.7) and was significantly lower with segments C2/C3 (0.3) and L2/L3 (0.19). Addition of a skeletal muscle target explant to the cord-SCG cocultures did not alter the preferential index of interaction between SCG and upper thoracic cord levels. Furthermore, the cholinergic cord neurons in medioventral strips did not promote increase of ChAT activity into equally accessible cocultured ganglia of inappropriate phenotype, e.g. sensory dorsal root ganglia. Decentralization of SCGs after coculture with appropriate T1/T2 cord resulted in loss of ganglionic ChAT activity. Electrical stimulation of the medial region in T1/T2 cord explants evoked compound ganglion action potentials in cocultured SCGs. The ganglion responses were blocked by hexamethonium. These data suggested that neurons located in the medial region of upper thoracic cord (presumably autonomic preganglionic) are able to develop enhanced cholinergic projections within cocultured SCGs, in comparison with neurons located in ventral cord (presumably motoneurons). In contrast, dorsal cord neurons showed no significant cholinergic interaction with SCGs. Furthermore, neurons located in upper thoracic spinal cord segments develop enhanced cholinergic projections within cocultured SCGs in comparison with neurons located in cervical and lumbar cord segments.     

c-ret regulates cholinergic properties in mouse sympathetic neurons: evidence from mutant mice

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c-ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss-of-function mutation of the c-ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate-limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT-positive cells are affected by the c-ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c-ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.     

Heterogeneous choline acetyltransferase staining in cholinergic neurons

We studied the features of choline acetyltransferase (ChAT) distribution in cholinergic structures of the central and peripheral nervous systems; specifically in the spinal cord and dorsal root ganglion (DRG) of embryo (E20), newborn, or adult rats using a goat polyclonal antibody. We found that this anti-ChAT antibody selectively labels both central and peripheral cholinergic structures. We observed cholinergic neurons with weak immunoreactivity. We demonstrated that at all stages of ontogeny that we studied, four groups of cholinergic neurons occurred in the cervical part of the spinal cord, viz., the small neurons of the dorsal horns, neurons of Rexed’s lamina X, neurons of the area of Rexed’s laminae VI–VII, and cells of the ventral horns. Interneurons of Rexed’s laminae VI–VII exhibited maximum ChAT reactivity as compared to other groups of cholinergic neurons of the spinal cord at all developmental stages we studied. We found an increase in the number of immunopositive cells of lamina X from E20 stage to the early postnatal and mature stages. In the peripheral cholinergic structures, i.e., the DRG, of the rat, all neurons expressed ChAT at all stages of ontogeny.     

Embryonic development of four different subsets of cholinergic neurons in rat cervical spinal cord

The developmental stage at which a neuron becomes committed to a neurotransmitter phenotype is an important time in its ontogenetic history. The present study examines when choline acetyltransferase (ChAT) is first detected within each of four different subsets of cholinergic neurons previously identified in the cervical enlargement of the spinal cord: namely, motor neurons, partition cells, central canal cluster cells, and dorsal horn neurons. By examining the temporal sequence of embryonic development of these cholinergic neurons, we can infer the relationships between ChAT expression and other important developmental events. ChAT was first detected reliably on embryonic day 13 (E13) by both biochemical and immunocytochemical methods, and it was localized predominantly within motor neurons. A second group of primitive-appearing ChAT-positive cells was detected adjacent to the ventricular zone on E14. These neurons seemed to disperse laterally into the intermediate zone by E15, and, on the basis of their location, were tentatively identified as partition cells. A third group of primitive ChAT-immunoreactive cells was detected on E16, both within and around the ventral half of the ventricular zone. By E17, some members of this "U"-shaped group appeared to have dispersed dorsally and laterally, probably giving rise to dorsal horn neurons as well as dorsal central canal cluster cells. Other members of this group remained near the ventral ventricular zone, most likely differentiating into ventral central canal cluster cells. Combined findings from the present study and a previous investigation of neurogenesis (Phelps et al.: J. Comp. Neurol. 273:459-472, '88), suggest that premitotic precursor cells have not yet acquired the cholinergic phenotype because ChAT is not detectable until after the onset of neuronal generation for each of the respective subsets of cholinergic neurons. However, ChAT is expressed in primitive bipolar neurons located within or adjacent to the germinal epithelium. Transitional stages of embryonic development suggest that these primitive ChAT-positive cells migrate to different locations within the intermediate zone to differentiate into the various subsets of mature cholinergic neurons. Therefore, it seems likely that spinal cholinergic neurons are committed to the cholinergic phenotype at pre- or early migratory stages of their development. Our results also hint that the subsets of cholinergic cells may follow different migration routes. For example, presumptive partition cells may use radial glial processes for guidance, whereas dorsal horn neurons may migrate along nerve fibers of the commissural pathway. Cell-cell interactions along such diverse migratory pathways could play a role in determining the different morphological, and presumably functional, phenotypes expressed by spinal cholinergic neurons.     

Development of cholinergic and GABAergic neurons in the rat medial septum: Different onset of choline acetyltransferase and glutamate decarboxylase mRNA expression

In the present study, we have investigated the developmental expression of the transmitter-synthesizing enzymes choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in rat medial septal neurons by using in situ hybridization histochemistry. In addition, we have employed immunostaining for ChAT and the calcium-binding protein parvalbumin, known to be contained in septohippocampal GABAergic neurons. A large number of GAD67 mRNA-expressing neurons were already observed in the septal complex on embryonic day (E) 17, the earliest time point studied. During later developmental stages, there was mainly an increase in the intensity of labeling. Neurons expressing ChAT mRNA were first recognized at E 20, and their number slowly increased during postnatal development of the septal region. The adult pattern of ChAT mRNA-expressing neurons was observed around postnatal day (P) 16. By using a monoclonal ChAT antibody, the first immunoreactive cells were not seen before P 8. Similarly, the first weakly parvalbumin-immunoreactive neurons were seen in the septal complex by the end of the 1st postnatal week. These results indicate that in situ hybridization histochemistry may be an adequate method to monitor the different development of transmitter biosynthesis in cholinergic and GABAergic septal neurons. Moreover, the late onset of ChAT mRNA expression would be compatible with a role of target-derived factors for the differentiation of the cholinergic phenotype. © 1996 John Wiley-Liss, Inc.     

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.     

Developmental change in the nerve growth factor action from induction of choline acetyltransferase to promotion of cell survival in cultured basal forebrain cholinergic neurons from postnatal rats

Nerve growth factor (NGF), a well-characterized target-derived growth factor, has been postulated to promote neuronal differentiation and survival of the basal forebrain cholinergic neurons. In the present paper, we demonstrate that a developmental change in NGF action occurs in postnatal rat basal forebrain cholinergic neurons in culture. Firstly, NGF acts as maturation factor by increasing choline acetyltransferase (ChAT) activity and acts later as a survival factor. In dissociated cell cultures of septal neurons from early postnatal (P1-4) rats, ChAT activities were increased by the addition of NGF. That is, ChAT activities in P1 septal cells cultured for 7 days was increased 4-fold in the presence of NGF at a concentration of 100 ng/ml. However, the number of the acetylcholinesterase (AChE)-positive neurons was not significantly different between these groups. In contrast, septal neurons from P8 to P14 rats showed different responses to NGF. Although the P14 septal neurons in culture for 7 days without NGF lost about half of the ChAT activity during a 7-day cultivation, cells cultured with NGF retained the activity at the initial level. The number of AChE-positive neurons counted in cultures with NGF was much greater than the number without NGF. These results suggest that, during the early postnatal days, the action of NGF on the septal cholinergic neurons in culture changes from induction of ChAT activity to the promotion of cholinergic neuronal cell survival. During this developmental period in vivo, septal neurons are terminating their projections to the hippocampal formation. Similar NGF-regulated changes in cholinergic neurons were observed in cultured postnatal neurons from vertical limb of diagonal band. An analogy has been pointed out between the neuronal death of the basal forebrain cholinergic neurons and a similar neuronal death in senile dementia, especially Alzheimer's type. The work reported here might present a possibility that NGF could play a role in preventing the loss of the basal forebrain cholinergic neurons in this disease.     

Chronic intraventricular injections of nerve growth factor elevate hippocampal choline acetyltransferase activity in adult rats with partial septo-hippocampal lesions

Nerve growth factor (NGF) was injected intraventricularly during 4 weeks into adult rats with unilateral partial lesions of the cholinergic septo-hippocampal pathway. On the lesioned side, NGF treatment elevated choline acetyltransferase (ChAT) activity up to 60% above the activity measured on the lesioned side of cytochrome c-treated controls. On the unlesioned side, NGF treatment increased ChAT activity only to an insignificant degree. ChAT activity in the septum of NGF-treated animals was increased by 60% as compared to controls. The NGF-induced increases on the lesioned side and in the septum were not accompanied by elevations in acetylcholinesterase (AChE) activity. Furthermore, histochemical analysis revealed no difference in AChE staining pattern or intensity between NGF-treated and control animals. The lack of effect on AChE strongly suggests that the increases in ChAT activity in hippocampus and septum are due to an elevation of ChAT activity within cholinergic neurons surviving the lesion rather than to a promotion of sprouting of cholinergic fibers.     

Choline acetyltransferase and substance P-like immuno-reactivity in the human spinal cord: changes in amyotrophic lateral sclerosis

The topographic location of the enzyme choline acetyltransferase (ChAT) has recently been determined within the human spinal cord². ChAT, which is regarded as a specific marker of cholinergic structures in nervous tissue, showed an area of high activity in the ventrolateral part of the ventral horn, probably related to motor neurons. In addition, an area of high ChAT activity was found in the apical part of the dorsal horn. As amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of the cortico-spinal tracts and the lower motor neurons, we considered it of value to investigate the involvement of spinal cholinergic structures in this disorder. Substance P is regarded as the transmitter of incoming pain signals to the dorsal horn of the spinal cord, a subject recently reviewed by Marx¹⁰. As disturbed sensation of pain is not a symptom of ALS, there seemed reason to correlate the spinal concentration of this peptide with the activities of ChAT in ALS.     

Choline acetyltransferase immunohistochemical staining in the goldfish (Carassius auratus) brain: Evidence that the Mauthner cell does not contain choline acetyltransferase

In the hatchetfish, the Mauthner cell (M-cell) is thought to be cholinergic based on electrophysiological studies using cholinergic agents and on the localization of acetylcholinesterase (AChE) and alpha-bungarotoxin to M-cell-giant fiber synapses. Immunocytochemical studies have shown that mammalian and non-mammalian cholinergic neurons stain positive for choline acetyltransferase (ChAT), the enzyme responsible for synthesizing acetylcholine. We processed tissue from the goldfish (Carassius auratus) for the immunohistochemical detection of ChAT using the monoclonal antibody AB8 and the peroxidase-antiperoxidase procedure. ChAT immunoreactivity was found in selected areas of the goldfish brain including the cranial nerve nuclei and the ventral horn motoneurons of the spinal cord. Interestingly, the M-cell soma which stains positive for AChE was ChAT negative. This immunohistochemical evidence does not support cholinergic functioning of the Mauthner cell.     

Distribution of cholinergic neurons in the chick spinal cord during embryonic development. Comparison of ChAT immunocytochemistry with AChE histochemistry.

The location of cholinergic neurons was studied during the development of the chick embryo spinal cord. A comparison between choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry was performed. ChAT-positive neurons could be detected only from embryonic day 9 (E9) onwards by the FITC technique and from E12 onwards by the PAP technique. These neurons were located mainly in the medial and lateral motor columns in the ventral horn of the gray matter and some of them were observed in the intermediate region of the spinal cord. AChE-containing cell bodies were much more numerous than the ChAT immunoreactive ones and were distributed in the ventral horn of the gray matter, the intermediate gray region and mostly off the apical part of the dorsal horn. ChAT should provide a reliable and specific marker for cholinergic neurons.     

Stimulation of choline acetyltransferase by C3d, a neural cell adhesion molecule ligand

Septal cholinergic neurons project to the hippocampus and release acetylcholine, a neurotransmitter involved in learning and memory. The enzyme choline acetyltransferase (ChAT) is responsible for synthesizing acetylcholine. Promoting ChAT activity and acetylcholine release can lead to new treatments for neurodegenerative diseases with cholinergic deficits, such as Alzheimer's disease. We present evidence that the synthetic molecule C3d, which is a peptide mimetic of the neural cell adhesion molecule (NCAM), promotes ChAT activity in cultures of rat embryonic septal neurons. Our data demonstrate that ChAT activity triggered by C3d is dependent on the fibroblast growth factor receptor (FGFR) and the mitogen-activated protein kinase (MAPK) pathway. C3d did not affect the number of cholinergic neurons in culture, indicating that NCAM homophilic binding enhances ChAT activity, without affecting cholinergic cell survival. In conclusion, the NCAM mimetic peptide C3d promotes ChAT activity in septal neurons through FGFR and MAPK. These findings are relevant to the design of new strategies aimed at stimulating cholinergic function and improving cognition in disorders such as Alzheimer's disease.     

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.     

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.     

Origin of neuronal nitric oxide synthase (NOS)-immunoreactive fibers in guinea pig parasympathetic cardiac ganglia

This study was conducted to determine the origin(s) of neuronal nitric oxide synthase-immunoreactive (NOS-IR) fibers within guinea pig atrial whole-mount preparations containing the cardiac ganglia. Intrinsic NOS-IR cardiac neurons exhibited choline acetyltransferase (ChAT) immunoreactivity, indicating that they were cholinergic as well as nitrergic. Comparison of control versus 72-hour explant culture preparations indicated that most of the nitrergic fibers within cardiac ganglia were extrinsic. The extrinsic NOS-IR fibers were not IR for ChAT (marker of preganglionic parasympathetic neurons), tyrosine hydroxylase (marker of catecholaminergic sympathetic postganglionic axons), or calcitonin gene-related peptide (CGRP) (marker of afferent fibers). Separate NOS-IR and ChAT-IR neurons were present within medullary regions containing the cardiovascular regulatory nuclei (nucleus ambiguus and dorsal motor nucleus of the vagus), but no cells were found that exhibited both NOS immunoreactivity and ChAT immunoreactivity. The small size and location of the medullary NOS-IR neurons suggested they were probably interneurons. Only an occasional sympathetic postganglionic cell in the stellate ganglion complex exhibited NOS immunoreactivity. NOS-IR cells were present in dorsal root ganglia (thoracic 1-5), but these typically also exhibited CGRP immunoreactivity. NOS-IR cells were also present in the nodose ganglia, but only some exhibited CGRP immunoreactivity. We concluded that virtually all the extrinsic NOS-IR nerve fibers represented an afferent fiber input that was separate from the substance P (SP)/CGRP-containing population of sensory fibers. Furthermore, much of this NOS innervation is probably derived from the nodose ganglia.     

Generation patterns of four groups of cholinergic neurons in rat cervical spinal cord: a combined tritiated thymidine autoradiographic and choline acetyltransferase immunocytochemical study

This report examines the generation of cholinergic neurons in the spinal cord in order to determine whether the transmitter phenotype of neurons is associated with specific patterns of neurogenesis. Previous immunocytochemical studies identified four groups of choline acetyltransferase (ChAT)-positive neurons in the cervical enlargement of the rat spinal cord. These cell groups vary in both somatic size and location along the previously described ventrodorsal neurogenic gradient of the spinal cord. Thus, large (and small) motoneurons are located in the ventral horn, medium-sized partition cells are found in the intermediate gray matter, small central canal cluster cells are situated within lamina X, and small dorsal horn neurons are scattered predominantly through laminae III-V. The relationships among the birthdays of these four subsets of cholinergic neurons have been examined by combining 3H-thymidine autoradiography and ChAT immunocytochemistry. Embryonic day 11 was the earliest time that neurons were generated within the cervical enlargement. Large and small ChAT-positive motoneurons were produced on E11 and 12, with 70% of both groups being born on E11. ChAT-positive partition cells were produced between E11 and 13, with their peak generation occurring on E12. Approximately 70% of the cholinergic central canal cluster and dorsal horn cells were born on E13, and the remainder of each of these groups was generated on E14. Other investigators have shown that all neurons within the rat cervical spinal cord are produced in a ventrodorsal sequence between E11 and E16. In contrast, ChAT-positive neurons are born only from E11 to E14 and are among the earliest cells generated in the ventral, intermediate, and dorsal subdivisions of the spinal cord. However, all cholinergic neurons are not generated simultaneously; rather their birthdays are correlated with their positions along the ventrodorsal gradient of neurogenesis. The fact that large motoneurons and medium-sized partition cells are born before small central canal cluster and dorsal horn cells would appear to support the generalization that large neurons are generated before small ones. However, the location of spinal cholinergic neurons within the neurogenic gradient seems to be more importantly associated with the time of cell generation than somal size. For example, when large and small motoneurons located at the same dorsoventral spinal level are compared, both sizes of cells are generated at the same time and in similar proportions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transplantation of fetal and early postnatal rat septal cholinergic neurons cultured in serum-free and serum-containing medium with nerve growth factor

Fetal rat (E17-E19) septal neurons were cultured in a defined, serum-free medium for 6-8 days with or without nerve growth factor (NGF) and transplanted into the hippocampus or the surrounding ventricle of 28 adult rats denervated of its septal input by a fimbria-fornix transection. The cholinergic septal neurons, which were visualized by acetylcholinesterase (AChE) histochemistry, always survived in transplantation to the adult brains from nearly pure neuronal cultures. Although choline acetyltransferase (ChAT) activity of septal neurons in culture was greatly increased (5.59-fold) by the addition of NGF to the defined medium, this ChAT induction appeared to have little effect on the subsequent survival or growth of the septal neurons after transplantation. These results demonstrate that survival of cultured fetal septal cholinergic neurons following transplantation is not dependent upon the presence of NGF or serum- or glia-derived factors during the preliminary culture. Postnatal rat (P4) septal neurons cultured for 5 days in serum-containing medium with NGF were also successfully transplanted in one of 3 cases.