Biomedical subjects

R P Barber

Publications and source records attributed to R P Barber.

At least 19 recordsLinked to original sources

PubMed1998

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.

Animals↗

PubMed1996

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.

Animals↗

PubMed1993

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.

Animals↗

PubMed1993

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.

Animals↗

PubMed1992

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)

Animals↗

PubMed1991

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.

Acetylcholine↗

PubMed1991

Embryonic development of choline acetyltransferase in thoracic spinal motor neurons: somatic and autonomic neurons may be derived from a common cellular group.

This investigation focused on the relationship between neurotransmitter phenotype expression and rat motor neuron development, as studied with choline acetyltransferase (ChAT) immunocytochemical techniques. The development of two subclasses of motor neurons, somatic and autonomic efferents, was examined in the upper thoracic spinal cord. ChAT was first detected in a few neurons on embryonic day 12 1/2 (E12 1/2), and in numerous cells located in a single, ventrolaterally located column in the intermediate zone on E13. By E14, this group of ChAT-positive neurons was more intensely immunoreactive, and their axons could be traced to appropriate targets in developing somatic muscle and paravertebral sympathetic ganglia. During the E15-16 period, somatic and autonomic motor neurons separated into two distinct subgroups, with the latter cells being observed to translocate dorsally. By E17, these autonomic motor neurons reached their final positions in the midportion of the intermediate zone. The autonomic motor neurons were observed to extend transverse dendritic bundles across the spinal cord between E15-16, but evidence of the longitudinal bundles of sympathetic preganglionic dendrites was not observed until after birth. A recent study of cholinergic thoracic motor neurons found that both somatic and autonomic cells were generated synchronously during the E11-12 period (Barber et al., Soc Neurosci Abstr 15:588, 1989). In combination with the present results, these data indicate that no more than 1 1/2 days are necessary after motor neuron genesis before a few cells begin to express detectable levels of ChAT, and that no more than 2 days are required before large numbers express this marker of the cholinergic phenotype. Further comparisons of the present findings with those of previous investigations of the development of both somatic and autonomic motor neurons (Dennis et al., Dev Biol 81:266, 1981; Rubin, J Neurosci 5:685, 697, 1985) indicate that these cells contain ChAT at the time their axons are growing toward their respective peripheral targets 1 day before the time when physiological evidence of function is manifest. Furthermore, the present results suggest that both subclasses of motor neurons initially migrate together from the ventricular zone into a single motor column within the ventral intermediate zone, and that the autonomic neurons subsequently translocate dorsally. Thus, autonomic motor neurons appear to be an exception to the generalization that postmitotic neurons migrate directly from the germinal zone to their final positions within the central nervous system.

Animals↗

PubMed1990

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.

Animals↗

PubMed1989

Localization of Drosophila neurons that contain choline acetyltransferase messenger RNA: an in situ hybridization study.

In situ hybridization with radiolabeled complementary RNA (cRNA) probes was used to determine the location of the messenger RNA (mRNA) encoding choline acetyltransferase (ChAT) in Drosophila nervous system. Areas in the cell-rich cortical regions of the cerebrum and optic lobes hybridized with substantial concentrations of the probe. This contrasted with the cell-sparse neuropil areas where no significant concentrations of probe were observed. Although most of the cortical regions were substantially labeled, there were regions within all of the areas where labeling was sparse or nonexistent. For example in the lamina, even though the monopolar cell layer appeared to be heavily labeled, there were some neuronal profiles that were not associated with the probe. Moreover, the epithelial glia that form an arch of cell profiles subjacent to the monopolar cells were not labeled, nor were amacrine neurons in the apex of the lamina near the external optic chiasma. The highest concentration of probe (approximately 140 grains/400 microns2) was observed in the laminar monopolar cell region and the cerebral cortical rind. The next most heavily labeled region (approximately 90 grains/400 microns2) occurred over cortical cells of the medulla-lobula. In the peripheral nervous system, label over the antennal sensory neurons amounted to about 75 grains/400 microns2, and the retinular cell layer of the compound eye exhibited about 60 grains/400 microns2. The control probe did not hybridize in significant quantities in either cellular or noncellular regions. This study presents evidence that large numbers of Drosophila cortical and primary sensory neurons contain the messenger RNA necessary for the production of ChAT, the acetylcholine-synthesizing enzyme. Further, our findings provide baseline information for use in ontogenetic studies of cholinergic neurons in Drosophila, and they also provide normative data for studying the effects of mutant alleles at the Cha or Ace loci upon the transcription of ChAT messenger RNA.

Animals↗

PubMed1988

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)

Animals↗

PubMed1988

Dendritic development and preferential growth into synaptogenic fields: a quantitative study of Golgi-impregnated spinal motor neurons.

Branching patterns of dendrites may be modulated by the way in which dendritic growth cone filopodia come into initial synaptic relationships with afferent axons. This synaptotropic hypothesis of dendritic branching predicts that dendritic growth will be directed preferentially into regions containing numerous prospective presynaptic elements. The developing mouse spinal cord provides a natural experiment to test this prediction, because synapses are found exclusively within the marginal zones bordering the motor columns during the early (E11-14) period of synaptogenesis. During this time, therefore, most motor dendritic growth would be expected to be directed laterally or ventrally into the marginal zones, whereas internally directed growth should become more prevalent later, when synaptogenesis begins to take place within the intermediate zone, i.e., the motor columns proper. A computer-assisted three dimensional reconstruction system has been used to test these expectations in Golgi preparations of developing mouse (C57BL/6J) spinal cords ranging in age from E13 through P1. Mean dendritic lengths and branch densities are significantly greater for marginal zone dendrites than for intermediate zone dendrites at early ages (E13-14), but there are no significant differences in these measures at later stages of development (P0,1). These findings are interpreted as meaning that motor dendritic growth is initially biased into the marginal zone by synaptogenic afferents and that this preferential distribution is progressively lost as synapses develop within the intermediate zone to attract or to stabilize internally directed dendritic growth. Thus the findings of this study are consistent with predictions of the synaptotropic hypothesis of dendritic branching.

Animals↗

PubMed1986

Differentiation of dorsal root ganglion cells with processes in their synaptic target zone of embryonic mouse spinal cord: a retrograde tracer study.

The differentiation of dorsal root ganglion (DRG) cells with central processes in their synaptic target zones was studied in the developing spinal cord of embryonic mice (C57BL/6J). On embryonic days 12-15 (E12-E15), horseradish peroxidase (HRP) was pressure injected into the intermediate region of developing spinal grey matter and the embryos were cultured in an oxygenated medium to allow retrograde HRP transport to the dorsal root ganglia. Labelled DRG cells were measured and classified into four categories representing successive developmental stages: primitive bipolar cells, early transitional bipolar neurons, late transitional bipolar cells, and pseudounipolar neurons. In the period between E12 and E15, retrogradely labelled DRG cells became larger and less elongated as a population. Furthermore, a quantitative analysis of the cell types labelled on successive embryonic days of injection indicated an increase in the relative morphological maturity of labelled cells. On E12, the labelled cell population consisted solely of primitive bipolar and early transitional bipolar cells whereas the more mature late transitional bipolar and pseudounipolar neurons were predominant at E15, with the change between these two relationships occurring between E14 and E15. The principal finding of this study was that even the less differentiated forms of DRG neurons had axons within their central synaptic target fields during embryonic stages of spinal cord development.

Animals↗

PubMed1986

An improved method of toluidine blue counterstaining of neurons in immunocytochemical preparations.

Immunocytochemical methods can identify individual neurons and processes made immunoreactive by virtue of the antigens they contain. However, frequently it is also useful to visualize surrounding nonimmunoreactive cells, but immunocytochemical procedures often interfere with the quality of subsequent counterstaining. This report describes an improved method of counterstaining immunocytochemical specimens with either aged (at least 1 year) or concentrated solutions of toluidine blue. This technique combines well with immunocytochemical preparations of at least two antigens, i.e., choline acetyltransferase and glutamic acid decarboxylase, to delineate nonimmunoreactive somata. Additionally, a method of photographing these color preparations is described that, by the use of an appropriate filter, allows one to illustrate sections essentially with and without blue counterstain in black and white photomicrographs.

Animals↗

PubMed1984

The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cord: an immunocytochemical study.

A monoclonal antibody to choline acetyltransferase (ChAT), the acetylcholine (ACh)-synthesizing enzyme, has been used to localize ChAT within neurons in immunocytochemical preparations of adult rat spinal cord. Morphological details of known cholinergic spinal neurons are presented in this study, and previously unidentified ChAT-containing neurons are also described. Immunoreaction product was present within cell bodies, dendrites, axons, and axon terminals, thereby allowing comprehensive descriptions of the distribution of ChAT-positive neurons and the interrelationships of their processes. In the ventral horn, ChAT-positive motoneurons were located in the medial, central, and lateral motor columns, and their dendrites formed elaborate longitudinal and transverse ChAT-positive bundles. These bundles were present throughout the rostrocaudal extent of the spinal cord. In the central gray matter, small ChAT-positive cell bodies were clustered around the central canal. Small longitudinal fascicles of immunoreactive processes were also observed in this region adjacent to the ependymal layer. The intermediate gray matter of virtually the entire spinal cord was spanned by medium to large ChAT-positive multipolar cells termed partition neurons. At autonomic spinal levels, partition neurons were intermingled with other immunoreactive cells that were identified as preganglionic sympathetic or parasympathetic neurons because of their locations and morphological characteristics. In the sympathetic system, four groups of ChAT-positive neurons were observed; the principal intermediolateral nucleus (ILp) in the lateral horn, the central autonomic cell column (CA) dorsal to the central canal, the intercalated nucleus (IC) located between ILp and CA, and the funicular intermediolateral neurons (ILf) in the white matter lateral to the ILp. The dendrites of ILp and CA neurons formed substantial longitudinal bundles within each group, as well as transverse bundles between the groups that resembled the rungs of a ladder. ChAT-positive cell bodies were also present in the dorsal horn, and those located in laminae III-V extended dendrites dorsally into a longitudinal plexus within lamina III.

Animals↗

PubMed1984

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.

Aging↗

PubMed1984

Immunocytochemical localization of glutamic acid decarboxylase in the dorsal lateral vestibular nucleus: evidence for an intrinsic and extrinsic GABAergic innervation.

Immunocytochemical methods were used to localize the gamma-aminobutyric acid (GABA) synthesizing enzyme glutamic acid decarboxylase (GAD) in normal and partially deafferented dorsal lateral vestibular nucleus (dLVN) of the rat. Normal dLVN exhibited more intense staining than the ventral part of the nucleus (vLVN), and this was mainly due to more numerous GAD+ axon terminals surrounding dLVN somata and within neuropil. Small GAD+ somata were also present. Electron microscopy suggested the existence of two types of GAD+ synaptic terminals. The major type conformed to descriptions of Purkinje cell terminals, whereas the minor type did not. Ablation of the anterior cerebellar vermis resulted in a 73% reduction in the number of GAD+ terminals, the percentage decrease being greater around neuronal somata (83%) than within neuropil (70%). The presence of GAD+ somata within the nucleus and the persistence of some GAD+ terminals after extensive lesions of the Purkinje cell afferents suggest that both extrinsic and intrinsic GABAergic neurons may contribute to information processing within the dLVN.

Afferent Pathways↗

PubMed1984

Species-specific second antibodies reduce spurious staining in immunocytochemistry.

Spurious staining related to the second (linking) antibodies was observed in immunocytochemical specimens processed with an unlabeled antibody method. Some of this staining was suspected to result from species cross-reactivity of the second antibodies with endogenous immunoglobulin Gs in the tissue. Therefore, species-specific second antibodies were obtained, and the staining patterns of tissue processed with such antibodies were compared with those of tissue processed with standard (nonspecies-specific) second antibodies. In these studies, a monoclonal antibody to choline acetyltransferase (ChAT) was utilized as the primary antibody, and a similarly prepared monoclonal antibody that did not react with ChAT served as a control antibody. Spurious staining that included staining of discrete tissue and cellular components as well as amorphous background staining was present in both control and experimental tissue processed with standard second antibodies. Such staining was virtually eliminated in tissue processed with species-specific second antibodies. In specimens from the central nervous system, for example, species-specific second antibodies greatly reduced dark staining within the area postrema, in the pia-arachnoid membranes, and around blood vessels as well as the staining of small dot-like structures within some large neurons. In addition, the general level of background staining was reduced in both adult and developing tissues, thus permitting clearer visualization of many positively stained structures. In peripheral tissues such as skeletal muscle, spurious staining of connective tissue elements was eliminated, allowing the observation of previously occluded ChAT-positive structures such as nerve fibers and motor end-plates. Thus, species-specific second antibodies appear to be very useful for immunocytochemistry, particularly when the primary antibody and the tissue to be studied are from closely related species.

Animals↗

PubMed1983

Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase.

Choline acetyltransferase (ChAT), the acetylcholine (ACh) synthesizing enzyme, has been localized immunocytochemically with a monoclonal antibody in light and electron microscopic preparations of rat central nervous system (CNS). The antibody was an IgG1 subclass immunoglobulin that removed ChAT activity from solution. The specificity of the antibody and immunocytochemical methods has been confirmed by the demonstration of ChAT-positive neurons in a number of well-characterized cholinergic systems. For example, ChAT-positive reaction product was present in the cell bodies of spinal and cranial nerve motoneurons, as well as in their axons and terminations as motor end-plates in skeletal muscle. In addition, the somata of preganglionic sympathetic and parasympathetic neurons were ChAT-positive. The specificity of staining was further supported by a lack of reaction product in several groups of neurons thought to use neuroactive substances other than acetylcholine. No specific staining was observed in control specimens. The findings indicated that ChAT had an extensive intraneuronal distribution in many cholinergic neurons, being present in cell bodies, dendrites, axons and axon terminals. ChAT-positive somata were found in the medial septum and diagonal band, the medial habenula, and the basal nucleus of, the forebrain, 3 regions that are sources of cholinergic afferents to the hippocampus, interpeduncular nucleus and cerebral cortex, respectively. In addition, positively stained cell bodies were present within the cerebral cortex. ChAT-positive punctate structures were observed in the ventral horn of the spinal cord, where electron microscopic studies demonstrated that some of these structures were synaptic terminals. Other regions containing numerous ChAT-positive puncta included the hippocampus, the interpeduncular nucleus and the cerebral cortex. The light microscopic appearance of these putative cholinergic terminals varied among different brain regions. Large punctate structures related to well-defined post-synaptic elements were characteristic of some regions, such as the ventral horn of the spinal cord, while smaller punctate structures and varicose fibers with a diffuse pattern of organization distinguished other regions, such as the cerebral cortex.

Animals↗