Treatment of refractory ITP and Evans syndrome by haematopoietic cell transplantation: is it indicated, and for whom?

Several lines of therapy have been established for patients with immune thrombocytopenia (ITP) and Evans syndrome. However, these therapies generally require prolonged administration, lead to profound immunosuppression and increased infectious risk, and are often poorly tolerated. While most patients with these disorders will respond to first-line steroid therapy, others will prove refractory or intolerant to multiple treatments. In these patients (and possibly even selected patients who are not considered refractory), autologous or allogeneic haematopoietic stem cell transplantation (HCT) may provide definitive therapy. We review the literature on the treatment of ITP and Evans syndrome with HCT and discuss its use in the management of these disorders. We also pose, for the purpose of discussion, research questions that will be important to address if HCT is to be considered a viable option for more patients with these diseases.

Development of cholinergic terminals around rat spinal motor neurons and their potential relationship to developmental cell death.

Neuron death seems to be regulated mainly by postsynaptic target cells. In chicks, nicotinic antagonists such as alpha-bungarotoxin (alphaBT) can prevent normal cell death of somatic motor neurons (SMNs). For this effect, however, alphaBT could be acting at peripheral neuromuscular junctions and/or central cholinergic synapses. To investigate this issue, we first studied the development of cholinergic terminals in the rat spinal cord by using vesicular acetylcholine transporter immunocytochemistry. Labeled terminals were seen in the ventral horn as early as embryonic day 15 (E15), the beginning of the cell death period. Thus, central cholinergic synapses form at the correct time and place to be able to influence SMN death. We next added alphaBT to organotypic, spinal slice cultures made at E15. After 5 days in vitro, the number of SMNs in treated cultures was substantially greater than in control cultures, indicating that alphaBT can reduce SMN cell death in rats as it does in chicks. Moreover, peripheral target removal led to extensive loss of SMNs, and such a loss occurred even in the presence of alphaBT, indicating the necessity of peripheral target for the alphaBT effect. Finally, to determine whether central cholinergic terminals also may be involved in SMN death, we delayed the alphaBT treatment until after central cholinergic terminals had disappeared from the slice cultures. The increased number of surviving SMNs, even in the absence of central terminals, argued that alphaBT acts at peripheral, not central, cholinergic synapses to rescue SMNs from developmental cell death.

Manipulation of intracellular calcium has no effect on rate of migration of rat autonomic motor neurons in organotypic slice cultures.

Migration of neurons is a key step in the formation of the central nervous system, and an increase in internal Ca(2+) concentration has been shown to increase the rate of migration of granule cells along radial glial processes in slices of postnatal cerebellum. In embryonic spinal cord, the non-radial migration of autonomic motor neurons from the ventral horn dorsally into the region of the intermediolateral nucleus differs from that of granule cells, so it is possible that the role of Ca(2+) may also differ in the migration of these two types of neurons. To investigate this possibility, we made organotypic slice cultures of thoracic spinal cord from rat embryos. In control slices after about one day in vitro, diaphorase-positive autonomic motor neurons had migrated 100 microm at a rate of 3.6 microm/h. In experimental slice cultures, we added pharmacological reagents that are known to either increase or decrease internal Ca(2+) levels, including some reagents used successfully in the aforementioned granule cell studies. None of the nine reagents had a significant effect on migration speed of autonomic motor neurons in slice cultures. Our results suggest that autonomic motor neuron migration is not regulated by internal Ca(2+) levels, and hence this mechanism may not be used universally by all types of neurons.

Peripheral and central target requirements for survival of embryonic rat dorsal root ganglion neurons in slice cultures.

Developmental cell death in the nervous system usually is controlled by the availability of target-derived trophic factors. It is well established that dorsal root ganglia (DRG) neurons require the presence of their peripheral target for survival, but because of their central projections, it is possible that the spinal cord also may be required. Before examining this possibility in rat embryos, we first used terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) to determine that thoracic DRG cell death occurred from embryonic day 15 (E15) to E18. To determine the target requirements of DRG neurons, we used organotypic slice cultures of E15 thoracic trunk segments. After peripheral target removal, essentially all DRG neurons disappeared within 5 d. In contrast, after removal of the spinal cord, approximately half of the DRG neurons survived for at least 8 d. Hence, some E15 DRG neurons could survive without the spinal cord. However, those DRG neurons that died after spinal cord ablation apparently required trophic factors from both central and peripheral targets, because the presence of only one of these tissues was not adequate by itself to support this cell group. Addition of neurotrophin-3 (NT-3) to the culture medium rescued some DRG neurons after CNS removal, suggesting a possible role for NT-3 in vivo. In other experiments, cultures were established from older (E16) embryos, and essentially all neurons survived after spinal cord ablation, even without added factors. These and other experiments indicated that approximately 65% of DRG neurons are transiently dependent on the CNS early in development.

Autonomic motor neuron migration and expression of nicotinamide adenine dinucleotide phosphate reduced diaphorase are dependent upon peripheral target.

Interactions of developing neurons with their postsynaptic targets play a significant role in neuronal differentiation. The goal of the present study was to determine if target contact affected the migration or differentiation of autonomic motor neurons (AMNs) in developing rat spinal cord. The peripheral targets of AMNs were excised microsurgically from histotypic spinal slices before the arrival of AMN axons. The migration of AMNs was assessed in DiI retrogradely labeled preparations, and the differentiation of these cells was evaluated by beta-nicotinamide adenine dinucleotide phosphate reduced diaphorase (NADPH-d) histochemistry. In target-deprived specimens, NADPH-d expression in AMNs was virtually eliminated. In addition, DiI-labeled AMNs were scattered throughout the intermediate spinal gray matter instead of being aggregated in the intermediolateral nucleus as in control slices. This observation indicated that migration of AMNs had occurred, but that it had been disorganized significantly by target removal on embryonic day 13 (E13). In sham, "incision-only" specimens from which peripheral target tissue was not removed, AMNs expressed NADPH-d and migrated normally, indicating that axotomy alone was not sufficient to disrupt AMN development. Previous studies have shown that target removal after the arrival of AMN axons at their postsynaptic targets on E14 has no affect on the organized migration of AMNs (Barber et al. [1993] J. Neurosci. 13:4898-4907). This observation together with the present results indicate that initial target contact is necessary for both the differentiation and directed migration of AMNs, and that this contact does not need to be sustained for these developmental events to progress normally.

Differences in developmental cell death between somatic and autonomic motor neurons of rat spinal cord.

Considerable knowledge concerning developmental cell death has come from the study of somatic motor neurons (SMNs), but a related set of spinal neurons, the autonomic motor neurons (AMNs), have been studied less extensively in this respect. In the present study, we used three different approaches to determine the amount of AMN cell death during normal development in the rat. First, target dependency was studied in organotypic slice cultures, and it was found that AMNs survived for at least 12 days after removal of their postsynaptic targets. No factors were added to the serum-free medium to substitute for the ablated targets, indicating that AMNs were able to survive without target-derived trophic factors. Such target-independent survival is not characteristic of neurons that undergo typical developmental cell death. Second, AMNs were counted in double-stained choline acetyltransferase immunocytochemical and NADPH diaphorase histochemical preparations at ages (postnatal days 4-22) encompassing the period when AMN postsynaptic target cells undergo developmental death. Neuron numbers were essentially identical at all ages examined, indicating that no AMN cell death occurred postnatally. Finally, from embryonic day 13 to postnatal day 22, animals were analyzed by using terminal transferase-mediated nick-end labeling to identify dying cells. Many fewer labeled cells were observed among AMNs than among SMNs. Thus, all three approaches indicated that there is a significant SMN/AMN difference in developmental cell death. The phenotypic trait(s) that underlies this difference may also be important in the relative resistance of AMNs to pathological conditions that induce death of SMNs, e.g., those involved in amyotrophic lateral sclerosis and excitotoxicity.

Differential vulnerability of autonomic and somatic motor neurons to N-methyl-D-aspartate-induced excitotoxicity.

Two closely-related subsets of spinal motor neurons are differentially vulnerable in the degenerative neurological disease amyotrophic lateral sclerosis. Autonomic motor neurons (i.e. preganglionic sympathetic neurons) survive in this disorder, whereas most spinal somatic motor neurons do not. The present study was undertaken in order to begin to understand the phenotypic differences between the two motor neuronal subsets which might contribute to this differential survival. Organotypic slice cultures of postnatal rat thoracic spinal cord were maintained in defined medium for one to 12 days in the presence or absence of N-methyl-D-aspartate or its antagonist, D-amino-phosphonopentanoic acid. Autonomic motor neurons that were stained for either nicotinamide adenine dinucleotide phosphate reduced diaphorase or choline acetyltransferase only were both able to tolerate 50 microM N-methyl-D-aspartate treatment for over seven days in culture with no apparent adverse effects. In contrast, cultures maintained for only one day in medium containing 50 microM N-methyl-D-aspartate showed a dramatic and highly significant decrease in the numbers of neurofilament-positive somatic motor neurons, as well as nicotinamide adenine dinucleotide phosphate reduced diaphorase-positive interneurons. These N-methyl-D-aspartate-induced effects were dose-dependent and blockable. The results of this investigation indicated that autonomic motor neurons and somatic motor neurons were differentially susceptible to N-methyl-D-aspartate-induced excitotoxicity, and that the resistance of autonomic motor neurons to this insult appeared to be independent of the nicotinamide adenine dinucleotide phosphate reduced diaphorase phenotype.

Differential vulnerability of two subsets of spinal motor neurons in amyotrophic lateral sclerosis.

The primary objective of this study was to determine the pattern of motor neuron loss in thoracic spinal cord from amyotrophic lateral sclerosis (ALS) patients. A prerequisite to this objective was to examine control human spinal cord with the techniques to be used for ALS specimens. Combined choline acetyltransferase (ChAT) immunocytochemistry and NADPH diaphorase histochemistry (a marker for nitric oxide synthase) revealed a staining pattern very similar to that seen in other mammals. Stained cell groups were present in the superficial dorsal horn (labeled only by diaphorase), the deep dorsal horn (double-labeled), the intermediate region (double-labeled), around the central canal (mostly double-labeled), autonomic motor neurons (AMNs; either double-labeled or ChAT-positive only), and somatic motor neurons (SMNs; ChAT-positive only). These similarities indicated that most cell types previously described in other mammals are present in human spinal cord. However, the percentage of AMNs that were double-labeled was much higher in humans (94%) than in rodents (approximately 66%) or in nonmammalian vertebrates (essentially 0%). In ALS, extensive loss of SMNs is known to occur in cervical and lumbar enlargements, and similarly, our specimens revealed a degeneration of nearly all SMNs in thoracic spinal cord. In contrast, the average number of AMNs in ALS specimens was not significantly different from that in controls, directly confirming clinical observations suggesting that AMNs do not degenerate in ALS. Most importantly, the percentage of AMNs that were diaphorase-negative was not decreased in ALS, indicating that AMN resistance in this degenerative neurological disorder probably is independent of nitric oxide synthase expression.

Nonradial migration of interneurons can be experimentally altered in spinal cord slice cultures.

During development, many migrating neurons are thought to guide on radially oriented glia to reach their adult locations. However, members of the 'U-shaped' group of cholinergic interneurons in embryonic rat spinal cord appeared to migrate in a direction perpendicular to the orientation of radial glia. This 'U-shaped' group of cells was located around the ventral ventricular zone on embryonic day 16 and, during the next two days, the constituent cells dispersed into the dorsal horn or around the central canal. During this period, these cells could be identified with either ChAT immunocytochemistry or NADPH-diaphorase histochemistry and they appeared to be aligned along commissural axons, suggesting that such processes, rather than radial glia, might guide their migration. An organotypic spinal cord slice preparation was developed and utilized for three different experimental approaches to studying this migration. In the first experiments, slices of embryonic day 16 cervical spinal cord were cultured for one, two or three days, and a relatively histotypic dorsal migration of 'U-derived' cells could be inferred from these sequential cultures. A second set of experiments focused on the direct observation of dorsally directed migration in living spinal cord cultures. Embryonic day 16 slices were injected with a lipophilic fluorescent label near the dorsal boundary of the 'U-shaped' cell group and the dorsal movement of labeled cells was observed using confocal microscopy. These experiments confirmed the dorsal migratory pattern inferred from sequentially fixed specimens. A third experimental approach was to transect embryonic day 16 slice cultures microsurgically in order to disturb the migration of 'U-derived' cells. Depending upon the amount of ventral spinal cord removed, the source of cells was excised and/or their guidance pathway was perturbed. The number and position of 'U-derived' cells varied with the amount of ventral cord excised. If more than 400 microns was removed, no 'U-derived' diaphorase-labeled cells were present, whereas if only 200-300 microns was removed, the cultures contained such cells. However, in this instance, many of the 'U-derived' neurons did not move as far dorsally, nor did they display their characteristic dorsoventral orientation. When results from these three experiments are taken together, they provide strong evidence that nonradial neuronal migration occurs in developing spinal cord and that the 'U-derived' neurons utilize such a migration to move from their ventral generation sites to their dorsal adult locations.

Commissural fibers may guide cholinergic neuronal migration in developing rat cervical spinal cord.

The present investigation examines the role of intercellular relationships in the guidance of neuronal migration in embryonic rat cervical spinal cord. A "U-shaped" group of cholinergic neurons, was first detected on embryonic days (E) 15.5-16 surrounding the ventral proliferative zone. At these stages, no cholinergic cells were observed in the dorsal spinal cord, but by E17, many of the "U-shaped" group of cholinergic cells appeared to have translocated dorsally, to become the cholinergic dorsal horn cells seen in older animals. Between E16 and E17, these choline acetyltransferase (ChAT)-immunoreactive cells displayed primitive processes oriented dorsoventrally, suggesting migration along that axis. Two early forming substrates present in embryonic spinal cord have been implicated in the guidance of other populations of migrating neurons: glial cells organized in radial arrays and commissural axons aligned along the dorsoventral axis. Involvement of the commissural fibers with cholinergic cell migration seems more likely because the fibers and the translocation pathway have similar orientations. In double-labeling immunocytochemical studies of E15.5-17 spinal cord, some immature ChAT-containing neurons were directly adjacent to commissural fibers, as identified by SNAP/TAG-1 immunoreactivity. The temporal and spatial coincidence of developing cholinergic neurons and commissural axons is consistent with the hypothesis that these neurons could use commissural fibers as migratory guides. In addition, conventional electron micrographs were examined to determine if immature neuronal profiles were physically apposed to commissural axons. Immature neurons with leading and trailing processes oriented dorsally and ventrally, respectively, were embedded within and aligned along bundles of commissural fibers or along other similarly oriented neurons. This direct apposition of immature cells to the surfaces of commissural axons and other bipolar neurons is consistent with the hypothesis that the "U-shaped" group of cholinergic neurons may use commissural axons and other cohort neurons for guidance during their dorsal migration.

Transient and continuous expression of NADPH diaphorase in different neuronal populations of developing rat spinal cord.

Nitric oxide is a novel intercellular messenger whose role in neuronal development is not yet known. As a first step toward elucidating its developmental function, we examined the pattern of NADPH diaphorase histochemical staining, an indicator of the presence of nitric oxide synthase, in the rat spinal cord at pre and postnatal ages. Some types of neurons expressed diaphorase activity transiently during development. For example, a subset of somatic motor neurons, located in the ventrolateral corner of a few caudal segments of the cervical spinal cord, were diaphorase-positive beginning on E15, but gradually became diaphorase-negative by birth. In contrast, other spinal neurons expressed diaphorase activity continuously from development into adulthood. Preganglionic autonomic motor neurons became diaphorase-positive early in their development, as they were migrating toward their adult positions. Other spinal neurons, such as those in superficial dorsal horn, first expressed diaphorase relatively late in their development, after reaching their final location. The transient expression in some cell types, as well as the early expression in others, suggest that nitric oxide may have an important role(s) during development, which may differ from its functions in the adult nervous system.

Choline acetyltransferase and NADPH diaphorase are co-expressed in rat spinal cord neurons.

Phenotypic diversity underlies the complex functioning of the nervous system. One characteristic in which neurons differ from one another is the kind of molecules that they use for intercellular signalling. The classical neurotransmitter acetylcholine, synthesized by the enzyme choline acetyltransferase, is used by five groups of neurons in the rat spinal cord. Another messenger is nitric oxide, which is synthesized by nitric oxide synthase. Neurons that express nitric oxide synthase can be stained specifically by NADPH diaphorase histochemistry. In the spinal cord, approximately five groups of neurons are labeled by the diaphorase reaction, and some of these populations overlap with cholinergic groups. To determine the proportions of neurons that co-express choline acetyltransferase and nitric oxide synthase, we performed choline acetyltransferase immunocytochemistry and diaphorase histochemistry on single sections of rat spinal cord. Some cell types were single-labeled: somatic motor neurons were choline acetyltransferase-immunoreactive only, and neurons in lamina II were diaphorase-positive only. Four cell groups included double-labeled cells. Autonomic motor neurons were either double-labeled (62%) or choline acetyltransferase-only (37%), partition cells in lamina VII were double-labeled (54%) or choline acetyltransferase-only (45%), neurons in laminae III-V of the dorsal horn were double-labeled (70%) or diaphorase-only (27%), and neurons surrounding the central canal were double-labeled (56%), choline acetyltransferase-only (23%) or diaphorase-only (21%). These data indicate that certain spinal cord populations may be heterogeneous with regard to the intercellular messenger phenotypes involving acetylcholine and nitric oxide.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient expression of beta-NADPH diaphorase in developing rat dorsal root ganglia neurons.

We report here that during early fetal rat development, most, if not all, dorsal root ganglion (DRG) cells were stained by the beta-NADPH diaphorase histochemical reaction, indicating that they expressed nitric oxide synthase. During late fetal development, most DRG cells lost their diaphorase activity, but a small subset (located in all DRGs, but primarily in mid-thoracic DRGs) remained diaphorase-positive in adult animals. The transient expression of this enzyme suggests that nitric oxide may play a role during development that differs from its function in mature cells.

Preganglionic autonomic motor neurons display normal translocation patterns in slice cultures of embryonic rat spinal cord.

Phenotypic differences between somatic and autonomic motor neurons (SMNs and AMNs, respectively) may be modulated by epigenetic factors during the histogenic migrations of these cells. In order to study this problem experimentally, we have developed an in vitro, organotypic slice preparation of embryonic rat spinal cord. Our main objectives for this preparation were to determine whether in vivo patterns of motor neuronal translocations were mimicked in vitro, and, if they were, to begin to analyze such movements with experimental procedures that cannot be applied to the study of mammalian spinal cord development in vivo. Using a modification of existing organotypic slice procedures, we have shown that ChAT, an axonal surface glycoprotein and a low-molecular-weight neurofilament protein are expressed in slices cultured for up to 21 d, thus indicating that spinal neurons remained viable in vitro for relatively long periods. Most importantly, retrograde labeling and subsequent confocal microscopy have shown that the SMNs and AMNs of the slice preparations become segregated ventrodorsally into two distinct subcolumns as seen in vivo. The formation of separate AMN and SMN subcolumns appears to result from a dorsal translocation of AMNs. The fact that this cellular movement occurs in the slice preparation has allowed us to follow this phenomenon directly within the same specimen over a period of days. In addition, we have been able to observe the translocation of AMNs following the removal of their peripheral synaptic targets. The results of these experiments provide further evidence that AMNs undergo a dorsal translocation during the course of spinal cord development, and that this cellular movement may be due to an active migration. They also indicate that AMN movement is not dependent upon continual connection of these neurons with the paravertebral sympathetic ganglia.

Embryonic development of rat sympathetic preganglionic neurons: possible migratory substrates.

Spinal somatic and autonomic (sympathetic preganglionic) motor neurons are generated synchronously and, subsequently, migrate from the ventricular zone together to form a common primitive motor column. However, these two subsets of motor neurons ultimately express several phenotypic differences, including somal size, peripheral targets, and spinal cord locations. While somatic motor neurons remain ventrally, autonomic motor neurons (AMNs) move both dorsally and medially between embryonic days 14 and 18, when they approximate their final locations in spinal cord. The goal of the present investigation was to determine the potential guidance substrates available to AMNs during these movements. The dorsal translocation was studied in developing upper thoracic spinal cord, because, at this level, the majority of AMNs are located dorsolaterally. Sections were double-labeled by ChAT (choline acetyltransferase) and SNAP/TAG-1 (stage-specific neurite associated protein/transiently expressed axonal surface glycoprotein) immunocytochemistry to visualize motor neurons and the axons of early forming circumferential interneurons, respectively. Results showed that during the developmental stage when AMNs translocated dorsally, SNAP/TAG-1 immunoreactive lateral circumferential axons were physically located along the borders of the AMN region, as well as among its constituent cells. These findings indicate that lateral circumferential axons, as well as the SNAP/TAG-1 molecules contained upon their surfaces, are in the correct spatial and temporal position to serve as guidance substrates for AMNs. The medial translocation was studied in developing lower thoracic-upper lumbar spinal cord, because, at this level, more than half of the AMNs are medially located. Sections were double-labeled by ChAT and vimentin immunocytochemistry to visualize motor neurons and radial glial fibers, respectively. Observations on consecutive developmental days of the medial translocation revealed that AMNs were aligned with parallel arrays of radial glial fibers. Thus, the glial processes could serve as guides for the AMN medial movement. Future experimental analyses will examine whether circumferential axons and radial glial fibers are in fact functioning as migratory guides during AMN development, and, if so, whether specific surface molecules on these guides trigger the subsequent differentiation of AMNs.

Association interneurons of embryonic rat spinal cord transiently express the cell surface glycoprotein SNAP/TAG-1.

SNAP/TAG-1 is a 135 kDa glycoprotein of the immunoglobulin superfamily that is transiently expressed upon the surfaces of developing axons. In the embryonic rodent spinal cord, this molecule is expressed by motor neurons, dorsal root ganglion cells, and commissural neurons (Yamamoto et al.: J. Neurosci. 6:3576-3594, 1986; Dodd et al.: Neuron 1:105-116, 1988). The commissural cells are a subset of early-forming dorsal horn interneurons whose axons follow a circumferential course in the embryonic spinal cord. The axons of commissural neurons cross the developing ventral commissure to terminate on contralateral synaptic targets, whereas those of the other subset of circumferential cells, the association interneurons, remain on the same side of the spinal cord to form ipsilateral, terminal synaptic fields. The difference between the axonal trajectories of these two subsets of nerve cells raised the question of whether or not association interneurons would also express the SNAP/TAG-1 epitope and, if so, how would this expression be related to that of the commissural cells. Immunocytochemistry for SNAP/TAG-1 and choline acetyltransferase (ChAT) was used to answer these questions. The results indicated that association interneurons expressed SNAP/TAG-1 epitopes and that this expression began later and lasted longer than that of the commissural neurons. Other new findings of this study included the identification of a lateral subgroup of commissural fibers that expressed SNAP/TAG-1 later than their more medially located counterparts, and these lateral fibers were more pronounced in the thoracic spinal cord than at cervical levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Small cholinergic neurons within fields of cholinergic axons characterize olfactory-related regions of rat telencephalon.

Small immunoreactive cholinergic neurons were detected in the main and accessory olfactory bulbs of the rat with choline acetyltransferase immunocytochemistry. Such cells were also found in additional forebrain regions that received direct efferent innervation from the main olfactory bulb, such as the anterior olfactory nucleus, two subdivisions of the olfactory amygdala (nucleus of the lateral olfactory tract and anterior cortical nucleus), and the cortical-amygdaloid transition zone. Cholinergic neurons located in these olfactory-related regions were similar to each other morphologically and to those previously described by other investigators in the cerebral cortex, the hippocampus, and the basolateral amygdala. Somal measurements indicated that choline acetyltransferase-positive cells in olfactory-related regions were all essentially the same size, measuring 13-14 by 8-9 microns in major and minor diameters, respectively. In addition, these small cells were commonly bipolar in form with thin, smooth dendrites, and such characteristics have generally been associated with intrinsic, local circuit neurons in the forebrain. Depending on their location, however, these small cholinergic neurons differed from each other with regard to their frequency and dendritic orientation within planar sections. Choline acetyltransferase-immunoreactive cells in most cortical regions were relatively numerous and usually exhibited long, planar dendrites oriented perpendicularly to the pial surface. In contrast, dendrites of cholinergic neurons found in "cortical-like" regions (e.g. olfactory bulbs or nucleus of the lateral olfactory tract) were relatively sparse in number and appeared to be distinctly non-planar and randomly oriented. Despite these differences, the small choline acetyltransferase-positive cells had many features in common, including their distribution within forebrain regions that contained substantial terminal networks of choline acetyltransferase-positive axons thought to be derived primarily from the basal forebrain complex. In the rat, at least, the presence of small cholinergic interneurons within forebrain regions innervated by the large cholinergic projection neurons of the basal forebrain seems to be developing as a general principle of telencephalic organization. However, differences in both the size and the distribution of the terminal fields derived from each source imply a functional diversity between the intrinsic and extrinsic cholinergic systems of the forebrain.

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord.

The displacement of immature neurons from their place of origin in the germinal epithelium toward their adult positions in the nervous system appears to involve migratory pathways or guides. While the importance of radial glial fibers in this process has long been recognized, data from recent investigations have suggested that other mechanisms might also play a role in directing the movement of young neurons. We have labeled autonomic preganglionic cells by microinjections of horseradish peroxidase (HRP) into the sympathetic chain ganglia of embryonic rats in order to study the migration and differentiation of these spinal cord neurons. Our results, in conjunction with previous observations, suggest that the migration pattern of preganglionic neurons can be divided into three distinct phases. In the first phase, the autonomic motor neurons arise in the ventral ventricular zone and migrate radially into the ventral horn of the developing spinal cord, where, together with somatic motor neurons, they form a single, primitive motor column (Phelps P. E., Barber R. P., and Vaughn J. E. (1991). J. Comp. Neurol. 307:77-86). During the second phase, the autonomic motor neurons separate from the somatic motor neurons and are displaced dorsally toward the intermediate spinal cord. When the preganglionic neurons reach the intermediolateral (IML) region, they become progressively more multipolar, and many of them undergo a change in alignment, from a dorsoventral to a mediolateral orientation. In the third phase of autonomic motor neuron development, some of these cells are displaced medially, and occupy sites between the IML and central canal. The primary and tertiary movements of the preganglionic neurons are in alignment with radial glial processes in the embryonic spinal cord, an arrangement that is consistent with a hypothesis that glial elements might guide autonomic motor neurons during these periods of development. In contrast, during the second phase, the dorsal translocation of preganglionic neurons occurs in an orientation perpendicular to radial glial fibers, indicating that glial elements are not involved in the secondary migration of these cells. The results of previous investigations have provided evidence that, in addition to glial processes, axonal pathways might provide a substrate for neuronal migration. Logically, therefore, it is possible that the secondary dorsolateral translocation of autonomic preganglionic neurons could be directed along early forming circumferential axons of spinal association interneurons, and this hypothesis is supported by the fact that such fibers are appropriately arrayed in both developmental time and space to guide this movement.

Generation patterns of immunocytochemically identified cholinergic neurons at autonomic levels of the rat spinal cord.

The time at which a neuron is "born" appears to have significant consequences for the cell's subsequent differentiation. As part of a continuing investigation of cholinergic neuronal development, we have combined ChAT immunocytochemistry and [3H]thymidine autoradiography to determine the generation patterns of somatic and autonomic motor neurons at upper thoracic (T1-3), upper lumbar (L1-3), and lumbosacral (L6-S1) levels of the rat spinal cord. Additionally, the generation patterns of two subsets of cholinergic interneurons (partition cells and central canal cluster cells) were compared with those of somatic and autonomic motor neurons. Embryonic day 11 (E11) was the first day of cholinergic neuronal generation at each of the three spinal levels studied, and it also was the peak generation day for somatic and autonomic neurons in the upper thoracic spinal cord. The peak generation of homologous neurons at upper lumbar and lumbosacral spinal levels occurred at E12 and E13, respectively. Somatic and autonomic motor neurons were generated synchronously, and their production at each rostrocaudal level was virtually completed within a 2-day period. Cholinergic interneurons were generated 1 or 2 days later than motor neurons at the same rostrocaudal level. In summary, the birthdays of all spinal cholinergic neurons studied followed the general rostrocaudal spatiotemporal gradient of spinal neurogenesis. In addition, the generation of cholinergic interneurons also followed the general ventrodorsal gradient. In contrast, however, autonomic motor neurons disobeyed the rule of a ventral-to-dorsal progression of spinal neuronal generation, thus adding another example in which autonomic motor neurons display unusual developmental patterns.

Development of rostrocaudal dendritic bundles in rat thoracic spinal cord: analysis of cholinergic sympathetic preganglionic neurons.

Using monoclonal antibodies to choline acetyltransferase (ChAT) and glial fibrillary acidic protein (GFAP), we have analyzed the development of the dendritic bundles formed by cholinergic sympathetic preganglionic neurons (SPNs) in relationship to changes in the organization of glial fibers. In adult rat thoracic spinal cord, SPNs in the intermediolateral (IML) and central autonomic (CA) regions extend dendrites in both the mediolateral and rostrocaudal directions, forming a ladder-like pattern in horizontal sections of thoracic spinal cord. We report that, while the mediolateral dendrites form prenatally, the rostrocaudal dendritic bundles are not detected until at least a week later, during early postnatal life. The rostrocaudal dendrites develop rapidly during the first postnatal week, and achieve an adult-like pattern by postnatal day 14. The observed ontogenetic arrangements of dendritic bundles were correlated with the developing organization of astroglial processes with which they are intimately associated. While the appearance of mediolateral dendrites is consistent with the radial organization of glial in the embryonic spinal cord, the developmental time course of the rostrocaudal dendritic bundles coincides with the transformation of glial cells from this predominantly radial or transverse orientation to the randomly-oriented, stellate pattern of mature astrocytes. This temporal association suggests that ontogenetic changes in the organization of glial cells may contribute to the differential development of mediolateral and rostrocaudal dendritic patterns in the spinal cord.