Neurons containing cholecystokinin mRNA in the mammillary region: ontogeny and adult distribution in the rat.

1. The ontogeny and adult distribution of neurons containing cholecystokinin (CCK) mRNA in the premammillary and mammillary nuclei and supramammillary region of the rat brain were studied using hybridization histochemistry. 2. The earliest detection of CCK mRNA in the mammillary region was on E14, followed by a marked increase in transcript levels during the next 4 days, a time during which neurons in this region still divide. During the first 2 weeks of life, few changes in the levels of CCK transcripts were seen, and an adult-like pattern of expression was seen on the twenty-first day of life. 3. Low levels of transcripts were present in numerous neurons located in all divisions of the medial nucleus and in the posterior nucleus known to project ipsilaterally to the anteroventral and anteromedial thalamic nuclei. In contrast, none of the neurons in the lateral nucleus (projecting bilaterally to the anterodorsal thalamic nucleus) had detectable transcripts. 4. Many neurons in the supramammillary nucleus had low, moderate, or high levels of transcripts. Some nearby nuclei (such as the dorsal premammillary nucleus) had smaller numbers of neurons with low levels of CCK mRNA, whereas others (such as the ventral premammillary nucleus) had none.

Interstitial cells of the adult neocortical white matter are the remnant of the early generated subplate neuron population.

The postnatal fate of the first-generated neurons of the cat cerebral cortex was examined. These neurons can be identified uniquely by 3H-thymidine exposure during the week preceding the neurogenesis of cortical layer 6. Previous studies in which 3H-thymidine birthdating at embryonic day 27 (E27) was combined with immunohistochemistry have shown that these neurons are present in large numbers during fetal and early postnatal life within the subplate (future white matter), that they are immunoreactive for the neuron-specific protein MAP2 and for the putative neurotransmitters GABA, NPY, SRIF, and CCK. Here, the same techniques were used to follow the postnatal location and disappearance of the early generated subplate neuron population. At birth (P0), subplate neurons showing immunoreactivity for GABA, NPY, SRIF, or CCK are present in large numbers and at high density within the white matter throughout the neocortex, and the entire population can be observed as a dense MAP2-immunoreactive band situated beneath cortical layer 6. Between P0 and P401 (adulthood), the MAP2-immunostained band disappears so that comparatively few MAP2-immunoreactive neurons remain within the white matter. There is a corresponding decrease in the number and density of neurons stained with antibodies against neurotransmitters. In each instance, these neurons could be double-labeled by the administration of 3H-thymidine at E27, indicating that they are the remnants of the early generated subplate neuron population. The major period of decrease occurs during the first 4 postnatal weeks, and adult values are attained by 5 months. Within the white matter of the lateral gyrus (visual cortex), the density of immunostained neurons decreases dramatically: MAP2, 82%, SRIF, 81%, and NPY, 96%. While SRIF-immunoreactive neurons compose a nearly constant percentage of MAP2-immunoreactive neurons in the white matter between P0 (22%) and P401 (23%), those immunoreactive for NPY decline from 18 to 4%. These changes occur during the same period in which there is less than a twofold increase in white matter area. These observations indicate that the interstitial neurons of the adult neocortical white matter are the oldest neurons of the cerebral cortex since most if not all are derived from the subplate neuron population. In addition, a quantitative analysis suggests that the postnatal decline in subplate neuron density cannot be accounted for solely through dilution by differential growth of the white matter and most likely reflects an absolute decrease in subplate neuron number.(ABSTRACT TRUNCATED AT 400 WORDS)

From bird song to neurogenesis.

One of the dogmas of neurobiology has it that when nerve cells in the vertebrate brain die, they are not replaced by new ones. The author finds to the contrary. He has shown that when the adult canary needs to learn new songs, it does grow some new neurons. The finding could eventually lead to the discovery of ways to repair lesions in the human brain.

Development and differentiation of neuronal subsets in asexually reproducing Microstomum lineare. Immunocytochemistry of 5-HT, RF-amide and SCPB.

The development of immunoreactivity (IR) in the nervous system of asexually reproducing Microstomum lineare has been studied by a combination of simultaneous and double immunostaining with antisera to 5-HT and RF-amide, as well as with monoclonal antibodies to SCPB (molluscan small cardioactive peptide). Immunoreactivity appears in a distinct sequential order. 5-HT antigenicity in the postpharyngeal commissure indicates the initiation of the development of a new zooid. The development of a new brain and pharyngeal plexus always starts in connection to the parental nerve cords. Significantly different developmental patterns are observed for the IR to 5-HT and RF-amide, whereas IR to SCPB has the same localization as that to RF-amide, but appears both weaker and later during the development. Influences of the immunoreactive substances on the asexual reproduction and the feeding behaviour are discussed.

A combined in ovo-in vitro system for studies of volatile compounds on brain development: differential effects of carbon tetrachloride on neurones and astrocytes.

A method for studies of organic solvent effects on the development of neurones and astrocytes is described. Chick embryos in ovo were exposed to a low concentration of carbon tetrachloride vapours. After the exposure, primary cultures of neurones and astrocytes were prepared from the brain hemispheres of the embryos. Toxic effects of the solvent were analyzed in the cultures during the course of cellular differentiation as changes in cell growth and morphology, respiratory activity, and biochemical properties characteristic of each cell type. The development of neurones from embryos exposed from day 5 to day 8 to 25 p.p.m. of carbon tetrachloride was not affected by the exposure. On the other hand, growth rate and respiratory activity of astrocytes from embryos exposed from day 11 to day 14 to 25 p.p.m. of CCl4 were affected by the exposure, whereas no morphological changes or changes in the cyclic AMP levels could be observed. Consequently, low concentrations of carbon tetrachloride seem to affect the development of astrocytes rather than that of neurones in our experimental system.

Migration of young neurons in adult avian brain.

Neurons are born in the ventricular walls of the vertebrate central nervous system. From there, the young neurons migrate to their final destinations, where differentiation occurs. Neuronal migration has been described during the ontogeny of the avian and mammalian brain. Whereas in mammals most neurogenesis occurs during early development, in the adult avian forebrain wide-spread neurogenesis continues to occur. How do neurons born in adulthood reach their final destination? We report here that small elongated cells, born in the ventricular zone adjacent to the lateral ventricle, differentiate into mature neurons 20-40 days later, after migrating over distances of up to 5 mm. Migration rates are highest (28 micron h-1) when young neurons migrate through regions which are rich in radial glia. The adult vertebrate brain offers unique opportunities for studying factors that regulate neuronal migration, pathfinding and differentiation.

Development of hypothalamic neurons in intraventricular grafts: expression of specific transmitter phenotypes.

The anlages of the medial-basal hypothalamus (MBH), septopreoptic area (POA), Rathke's pouch, and the parietal cortex (CC) of rats (at 12.5, 14.5 and 16.5 days of gestation) were transplanted singly or in combination into the third ventricle of adult female rats, and the development of neurons in the grafts was investigated immunohistochemically with the use of antisera to tyrosine hydroxylase (TH), somatostatin (SRIH), ACTH, methionine enkephalin-Arg6-Gly7-Leu8 (Enk-8), rat corticotropin-releasing factor (rCRF), rat hypothalamic growth hormone-releasing factor (rhGRF), and luteinizing hormone-releasing hormone (LHRH). TH and all the peptides examined except LHRH were detected in distinct neurons in MBH grafts and in cografts of MBH plus Rathke's pouch from 12.5-day-old embryos. SRIH, rCRF, Enk-8, and TH were found in POA grafts from embryos of the same age. Although immunoreactive LHRH was first detected in neurons in POA grafts from 16.5-day-old embryos, it appeared in cografts of POA and MBH from 12.5-day-old embryos. The immunoreactive fibers developed in the grafts expressed the same characteristic behaviors as in intact brain; the fibers containing hormonal substances formed complexes with the vasculature like in the organum vasculosum laminae terminalis (OVLT) or in the median eminence, while the fibers containing neurotropic signals formed fiber networks surrounding other nerve cell bodies as if they synaptically associate. In CC grafts, the neurons contained TH, SRIH, rCRF, or Enk-8, and their axonal processes formed fiber networks. These findings suggest that all the hypothalamic neurons examined are committed by 12.5 days of gestation to develop maintaining transmitter phenotype and target recognition capacity.

Fetal antigen retained by mature neurons and ependyma studied with a monoclonal antibody (6B9).

A mouse monoclonal antibody (MAb 6B9, isotype IgM) was raised against autopsy tissue samples from the central nervous system (CNS) of multiple sclerosis (MS) patients. By immunofluorescence microscopy, MAb 6B9 intensely stains most or all cells in fetal rats. However, MAb 6B9 differentially stains various cell types in adult rats. Neurons, ependymal cells, and adrenal chromaffin cells are stained intensely, whereas astrocytes and oligodendrocytes are not stained. The 6B9-reactive antigen (6B9 antigen) is sensitive to periodic acid, but insensitive to treatment with protease, RNase, or hyaluronidase. Results from immunofluorescence microscopy on semithin sections and cultured neuroblastoma cells indicate that 6B9 antigen is intracellular. This is supported by immunoelectron microscopy, where labeling for 6B9 antigen appears in the cytoplasm distinct from any identifiable organelle. Further studies on 6B9 antigen should reveal its chemical nature as well as the significance of developmental changes in its distribution.

Development of voltage-dependent calcium, sodium, and potassium currents in Xenopus spinal neurons.

Action potentials of embryonic nerve and muscle cells often have a different ionic dependence and longer duration than those of mature cells. The action potential of spinal cord neurons from Xenopus laevis exhibits a prominent calcium component at early stages of development that diminishes with age as the impulse becomes principally sodium dependent. Whole-cell voltage-clamp analysis has been undertaken to characterize the changes in membrane currents during development of these neurons in culture. Four voltage-dependent currents of cells were identified and examined during the first day in vitro, when most of the change in the action potential occurs. There are no changes in the peak density of the calcium current (ICa), its voltage dependence, or time to half-maximal activation; a small increase in inactivation is apparent. The major change in sodium current (INa) is a 2-fold increase in its density. In addition, more subtle changes in the kinetics of the macroscopic sodium current were noted. The peak density of voltage-dependent potassium current (IKv) increases 3-fold, and this current becomes activated almost twice as fast. No changes were noted in the extent of its inactivation. The calcium-dependent potassium current (IKc) consists of an inactivating and a sustained component. The former increases 2-fold in peak current density, and the latter increases similarly at less depolarized voltages. The changes in these currents contribute to the decrease in duration and the change in ionic dependence of the impulse.

The development of a population of spinal cord neurons and their axonal projections revealed by GABA immunocytochemistry in frog embryos.

The development of a population of cerebrospinal-fluid-contacting neurons in the spinal cord of the Xenopus embryo ('Kolmer-Agduhr' cells) has been followed by using an immunocytochemical procedure that identifies GABA in fixed nervous tissue. Stained Kolmer-Agduhr cells containing GABA first appeared at stage 25 and their numbers increased steadily with the developmental age of the embryo. The Kolmer-Agduhr neurons had ascending ipsilateral axons that often terminated in growth cones. These axons and growth cones could be stained by the GABA antiserum from the earliest stages of outgrowth from the Kolmer-Agduhr cell body. We measured the angle of the earliest axons' outgrowth relative to the rostrocaudal axis of the spinal cord. The initial outgrowth of axons was always rostral over a narrow range of angles. This observation is inconsistent with the hypothesis of random initial outgrowth followed by later selection of the correct orientation, which would predict that axons would initially grow out over a wide range of angles. Instead, it suggests that, even from the earliest moments, axon outgrowth from the Kolmer-Agduhr cells is directed rostrally in a specific stereotyped manner.

Rapid regulation of neuronal growth cone shape and surface morphology by nerve growth factor.

Scanning electron microscopy was used to study regulation of growth cone shape and surface morphology by nerve growth factor (NGF). The growth cones of cultured rat sympathetic neurons and neuronally-differentiated PC12 cells were observed under conditions of continuous NGF exposure, NGF withdrawal, and NGF readdition. Growth cones of cells cultured in the continuous presence of NGF were mostly spread in shape and about 60% possessed surface ruffles. Ruffles appeared to be largely restricted to growth cones in that few were observed on cell bodies and neurites. Withdrawal of NGF for 4-5 hr caused most of the growth cones to take on a non-spread or contracted appearance and to lose their ruffles. Readdition of NGF promoted rapid changes in growth cone properties. Within 30 sec, ruffling was again evident on the growth cones and remained prominent there throughout the course of treatment (up to 5 hr). This was in contrast to cell bodies on which, as previously reported, ruffling also occurred following NGF readdition, but only transiently (for less than 15 min). Respreading of growth cones also occurred under these conditions. This was evident within 1 min of NGF readdition and reached the levels observed in continuously-treated cultures within 1-2 hr. Neurites were also examined. Ruffles were only rarely present in the continuous presence of NGF and were absent after NGF withdrawal. NGF readdition elicited ruffling along neurites within 30 sec; the prevalence of such ruffles diminished to that seen in continuously-treated cultures within about an hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal development of neurons containing both catecholaminergic and GABAergic traits in the rat main olfactory bulb.

Postnatal development of catecholaminergic and gamma-aminobutyric acid (GABA)-ergic neurons in the periglomerular region of the rat main olfactory bulb was studied immunohistochemically using antisera against tyrosine hydroxylase (TH), glutamic acid decarboxylase and GABA. TH-like immunoreactive neurons almost always contained GABA-like immunoreactivity in the first postnatal week, but about 10% of them did not contain GABA-like immunoreactivity in older animals.

Control of neuron shape during development and regeneration.

We have examined the ability of Mueller reticulospinal neurons in the CNS of the larval sea lamprey to sprout following axonal and dendritic injury. Axotomy induces regenerative sprouting exclusively from the axon stump if it occurs at a site distant from the soma in the spinal cord. However, axotomy within the hindbrain at a site close to the soma results in profuse neuritic sprouting from the dendrites. The gross morphology and trajectories of these "dendritic" sprouts resemble those of regenerating axons. Amputation of Mueller cell dendrites (dendrotomy) without axotomy does not result in neuritic sprouting from either the axon or dendrites, indicating that axotomy is specifically required for sprouting to occur. However, dendrotomy is capable of altering the distribution of sprouting in a previously axotomized Mueller cell by inducing sprouting at the site of the dendrotomy lesion. Sprouts of both dendritic and axonal origin tend to follow linear, rostrocaudally oriented paths along or near the ventral surface of the hindbrain. Some sprouts form very large, palmate growth cones on the marginal surface, which in turn give rise to many branches that continue to grow either rostrally or caudally along the surface of the brain. We discuss the possibility that both dendritic and axonal sprouts evoked by axotomy of Mueller neurons are recapitulating initial axonal development during embryogenesis, and that their trajectories are determined by developmental guidance cues persisting in the ventral hindbrain.

Assembly of microtubules at the tip of growing axons.

The growth of axons in the developing nervous system depends on the elongation of the microtubules that form their principal longitudinal structural element. It is not known whether individual microtubules in the axon elongate at their proximal ends, close to the cell body, and then move forward into the lengthening axon, or whether tubulin subunits are transported to the tip of the axon and assembled there onto the free ends of microtubules. The former possibility is supported by studies of slow axonal transport in mature nerves from which it has been deduced that microtubule assembly occurs principally at the neuronal cell body. By contrast, the polarity of microtubules in axons, which have their 'plus' or 'fast-growing' ends distal to the cell body, suggests that assembly occurs at the growing tip, or growth cone, of the axon. We have addressed this question by topically applying Colcemid (N-desacetyl-N-methylcolchicine), and other drugs which alter microtubule stability, to different regions of isolated nerve cells growing in tissue culture. We find that the sensitivity to these drugs is greatest at the growth cone by at least two orders of magnitude, suggesting that this is a major site of microtubule assembly during axonal growth.

Growth-associated protein, GAP-43, a polypeptide that is induced when neurons extend axons, is a component of growth cones and corresponds to pp46, a major polypeptide of a subcellular fraction enriched in growth cones.

Growth-associated protein, GAP-43, is a polypeptide that is induced in neurons when they grow axons. We show by means of subcellular fractionation and immunohistochemical localization that GAP-43 is a component of neuronal growth cones as well as growing neurites; it is similar to a major phosphoprotein, pp46, of a growth cone-enriched subcellular fraction. These conclusions are consistent with the possibility that the induction of GAP-43/pp46 is an important event in the establishment of a productive growth state in which a neuron is competent to extend an axon.

Effect of cytosine arabinoside on the composition of the nonneuronal cell population in cerebral explants.

Rat cortical explants were cultured in the presence or absence of the mitotic inhibitor cytosine arabinoside to determine whether or not it affects the composition of the nonneuronal cell population within the outgrowth zone. Ultrastructural morphometric analysis of the incidence of fibroblasts, fibrous astrocytes, and protoplasmic astrocytes-epithelial cells at 18 days in vitro, revealed statistically significant decreases in the incidence of fibroblasts and fibrous astrocytes in the explants treated with the inhibitor compared with control explants. Coupled with earlier findings of enhanced neurite outgrowth and decreased nonneuronal cell proliferation that follows such treatment, it appears that cytosine arabinoside may potentiate neurite outgrowth by altering the composition, as well as the number, of nonneuronal cells in the outgrowth zone. These data indicate that fibroblasts and fibrous astrocytes may limit the regenerative response of severed axons in the mammalian central nervous system.