Neuronal stimulation of [3H]thymidine incorporation by primary cultures of highly purified non-neuronal cells

A specific intercellular interaction has been demonstrated between neuronal and non-neuronal cells that appears to increase the rate of non-neuronal cell proliferation. Isolated and recombined primary cultures of both cell types were prepared from 11-day embryonic chick sympathetic ganglia by a method recently developed in this laboratory. When non-dividing neurons were added to an equal number of proliferating non-neuronal cells, the amount of [methyl-³H]thymidine incorporated by these mixed cultures was 230% greater than that incorporated by 99% pure non-neuronal cultures. Removal of all neurons from such non-neuronal cultures by a 48-h preincubation without nerve growth factor resulted in an even greater increase in [³H]thymidine incorporation upon addition of neurons (370%). When increasing numbers of isolated neurons were added to non-neuronal cell cultures, the amount of [³H]thymidine incorporation initially increased in a dose-dependent fashion until it reached a plateau. In contrast, the addition of increasing numbers of non-neuronal cells to a constant number of neurons resulted in a linear increase in [³H]thymidine incorporation. In some cases neurons and non-neuronal cells were not grown in direct physical contact but were only allowed to communicate with one another through the culture medium. Such indirect communication never resulted in a stimulation of [³H]thymidine incorporation. When neurons were added to cultures of embryonic chick fibroblasts, the neurons grew well but did not stimulate [³H]thymidine incorporation by the fibroblasts. These results suggest that embryonic sympathetic neurons selectively stimulate the proliferation of non-neuronal cells derived from the same source.     

Bromocriptine inhibits incorporation of [H]thymidine into rat pituitary tumor cells

The effects of bromocriptine on GH₃ pituitary tumor cell [³H]thymidine incorporation were studied. Cells were grown in the presence of bromocriptine, then exposed to a short-term pulse of [³H]thymidine in serum-free medium containing deoxycytidine (10 μM) to prevent deoxythymidine triphosphate (dTTP) pooling. After 48 h exposure to bromocriptine, basal prolactin (PRL)-secretion during 45 min was inhibited by 50% by 10 μM bromocriptine and thyroid releasing hormone-induced PRL stimulation was suppressed. Incorporation of radiolabelled thymidine into acid-precipitable DNA increased progressively from 15 to 60 min and was abolished by simultaneous incubation with excess unlabelled thymidine (100 μM). Bromocriptine (10 μM) inhibited incorporation of 5–50 μM [³H]thymidine, but this was not reversed by simultaneous incubation with metoclopramide (10 μM). Aminopterin, an inhibitor of endogenous de novo DNA synthesis, stimulated [³H]thymidine incorporation twofold and this increased DNA salvage pathway activity was also blocked by bromocriptine. As incorporation of [³H]thymidine into acid-soluble cell neucleotides was also inhibited by bromocriptine, the data suggest that in these cells the drug inhibits thymidine kinase activity, a salvage pathway of DNA synthesis.     

Autoradiographic evidence for receptor cell renewal in the olfactory epithelium of a snail

The tentacles of the terrestrial snail Achatina fulica contain an epithelium at their tips which is specialized for olfaction. The histology of the snail's olfactory organ bears a striking resemblance to that of the olfactory mucosa in the nose of vertebrates, where the receptor cell population is known to undergo a continuous process of renewal. In the present experiments, [³H]thymidine was delivered as a single pulse that was determined to have a maximum duration of about 1 h. Thirty minutes after an injection of [³H]thymidine, presumptive precursor cells were found labeled within, or at the edges of, receptor cell lobules. At later survival times, label was seen over cells that were identified as receptors. The mean position of the labeled cells within the layer of receptor cells became progressively more superficial with increasing survival times, indicating an upward migration of newly differentiated cells. The labeling index in the snail is ca. 0.7%, compared to 0.9% in the mouse. The turnover time is about 45 days, compared to 30–45 days in the mouse.     

Glial DNA synthesis and cell proliferation in the lesioned frontal cortex of the rat

Proliferation of rat neuroglial cells was quantified following a lesion of the frontal cortex, with the rate of incorporation of intraventricularly administered [³H]thymidine ([³H]TdR) into cortical DNA serving as an index of glial proliferation. Incorporation of [³H]uridine into the corresponding RNA fractions did not serve this purpose. The intraventricular route of administration of thymidine greatly reduced the amount of [³H]TdR needed to label DNA relative to systemic injection. The rate of incorporation of [³H]TdR into DNA was linear for 75 min post-injection. Significantly more [³H]TdR was incorporated into DNA of the lesioned frontal cortex than that of the contralateral control cortex, during the first 4 days post-trauma. The majority of the acid-insoluble radioactivity (from [³H]TdR) was localized in the nuclear subcellular fraction of the cortex. Experiments indicated that the enhanced incorporation of [³H]TdR was not the result of altered metabolism or pool sizes of TdR in the lesioned cortex. Histological analysis indicated that there was a significant increase in the number of glial cells in the lesioned cortex by day 4 post-lesion, which corresponded to the increase in DNA synthetic activity.It was concluded that mechanical trauma to the frontal cortex of the rat results in an increase in the number of glial cells at and near the lesion which is accompanied by an increase in incorporation of [³H]TdR into cortical DNA. This method of measuring posttraumatic DNA synthesis has several advantages over autoradiography.     

Selective sparing of later-born ganglion cells after neonatal transection of the infraorbital nerve

A combination of [³H]thymidine labelling and retrograde tracing with either horseradish peroxidase (HRP) or true blue (TB) was used to determine whether V primary afferent neurons born on different embryonic (E) days were differentially susceptible to neonatal transection of the infraorbital nerve (ION). In one experiment, rat fetuses were exposed to [³H]thymidine on E-8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, or 15.5, the left infraorbital nerve (ION) was transected on the day of birth, and both the regenerate and intact IONs were labelled with HRP when the animals reached adulthood. The percentage of HRP labelled cells that were also heavily labelled by [³H]thymidine was calculated for both the intact ganglion and that ipsilateral to the damaged nerve for each animal. A consistently higher percentage of double labelled cells on the lesioned rather than on the intact side for a given E-day was taken as an indication that cells born on the day in question had an increased probability of survival relative to the entire population of V ganglion cells that contributed axons to the ION. Cells born late in gestation on E-12.5 through 14.5 were significantly more likely than early born (E-9.5 through 11.5) cells to survive neonatal axotomy. In a second experiment, fetuses were exposed to [³H]thymidine on either E-9.5, E-10.5, or E-14.5, the vibrissa pads on both sides of the face were injected with TB within 6 hours of birth, and the ION was transected 6–8 hours later. When these rats reached at least 60 days of age, ganglia were processed for the visualization of both TB and [³H]thymidine labelled neurons. Cells labelled with both tracers would have been born on a given E-day, projected to the vibrissa pad via the ION at the time of nerve transection, and survived any naturally occurring or lesion-induced cell death. As in the HRP tracing experiment, ganglion cells born on E-14.5 were significantly more likely to survive neonatal ION transection than those born on either E-9.5 or E-10.5. © 1993 Wiley-Liss, Inc.     

Histogenesis of the vestibular sensory epithelium in organotypic culture of mouse embryo otocysts: A tritiated thymidine autoradiographic study

The cytogenesis in the vestibular sensory epithelium of mouse embryo otocysts grown in organ culture was examined using tritiated thymidine autoradiography. Pulse-labeling with [³H]-thymidine was applied either in vivo, at various intervals before explantation, or in vitro at specific stages of development. Observations of the development in vitro showed that the cytogenesis and cytodifferentiation of vestibular sensory cells were disturbed by explantation. By varying the intervals between the [³H]thymidine exposure and the date of explantation, we were able to demonstrate that explantation itself caused a significant decrease in the mitotic and the postmitotic phases of the hair cell precursors. Labeling of the expiants excised on day 13 of gestation revealed that precursors of the hair cells were progressively losing their mitotic capacity in vitro. In contrast with this finding, precursors of the supporting cells were less affected by explantation and culture conditions and they retained the capacity for survival, proliferation and differentiation.     

Development of hypothalamic opioid neurons: A combined immunocytochemical and [H]thymidine autoradiographic study

Using a combined technique of immunocytochemistry and [³H]thymidine autoradiography, we have determined the “birth-date” of opioid peptide containing neurons in three hypothalamic nuclei. These include proopiomelanocortin neurons (indicated by ACTH immunoreactivity) in the arcuate nucleus, dynorphin a neurons in the supraoptic nucleus, and [Leu]enkephalin neurons in the periventricular nucleus. Arcuate proopiomelanocortin neurons were born very early in embryonic development, with peak heavy [³H]thymidine nuclear labelling occurring on embryonic day E12. Supraoptic dynorphin A neurons were also labelled relatively early (peak at E13). By contrast, [Leu]enkephalin neurons in the periventricular nucleus exhibited peak heavy nuclear labelling on day E14. The results indicate a differential genesis of these three opioid peptide containing neuronal groups in three different hypothalamic nuclei.     

Insulin and the insulin-like growth factors I and II are mitogenic to cultured rat sciatic nerve segments and stimulate [3H]thymidine incorporation through their respective receptors

The factors that control proliferation of Schwann cells during peripheral nerve regeneration are not yet known. In this study we investigated the effects of insulin, insulin-like growth factor I and II (IGF-I and IGF-II), IGF-I analogues, and factors that interfere with their respective receptors, on [³H]thymidine incorporation into cultured nerve segments from the rat sciatic nerve. Segments cultured in nM (0.1–1.7 nM) concentrations of insulin, truncated IGF-I (tIGF-I), long R³IGF-I, or IGF-II exhibited an increase in [³H]thymidine incorporation compared with control segments. IGF-II was most potent. JB1, an IGF-I antagonist, counteracted the effects of tIGF-I and insulin. The results suggest that non-neuronal cells in the nerve segment, probably Schwann cells, possess distinct receptors for insulin, IGF-I, and IGF-II and that these receptors may be involved in the control of Schwann cell proliferation during peripheral nerve regeneration. © 1996 Wiley-Liss, Inc.     

Neuron loss in the mouse hippocampus following prenatal injection of tritiated thymidine or saline

To investigate possible effects of injections of tritiated thymidine ([3H]dThd) into pregnant mice or the injection procedure itself on the proliferation of neuronal precursor cells in the fetuses, pregnant mice received intraperitoneal injections of either [3H]dThd or saline on embryonic days 12, 14, and 19, while their offspring remained untreated. A second group of dams was not injected but their male offspring received a subcutaneous injection of again either [3H]dThd or saline on postnatal day 10. Then total numbers of hippocampal pyramidal cells (areas CA1 to CA3) and granular cells (dentate gyrus) were determined stereologically for 20-day-old as well as for 80-day-old male pups. No significant differences were found for the mean total number of pyramidal cells between the investigated groups of pups. However, the mean total number of granular cells was significantly reduced in those groups in which the dams had received an intraperitoneal injection, irrespective of whether [3H]dThd or saline was injected. This revives the repeated warning in the literature to consider the effect of the injection procedure on the developing brain when interpreting possible effects of agents administered during pregnancy.     

Experimental studies on the olfactory marker protein. II. Appearance of the olfactory marker protein during differentiation of the olfactory sensory neurons of mouse: an immunohistochemical and autoradiographic study

The time interval between the incorporation of [³H]thymidine and the appearaance of olfactory marker protien (OMP) in autoradiographically labeled neurons which have differentiated from stem cells, has been determined by autoradiographic and immunohistochemical techniques. The first [³H]thymidine-labeled, OMP-containing elements have been observed 7 days after administration of the radioactive thymidine. This result allows some speculation on the potential function of the olfactory marker protein.     

Regulation of Schwann cell proliferation in cultured segments of the adult rat sciatic nerve

Schwann cell proliferation was studied in cultured segments of the rat sciatic nerve by measurement of [³H] thymidine incorporation or through bromodeoxyuridine-(BrdU)-labelling and immunocytochemistry. The aim was to delineate mechanisms involved in the injury-induced proliferative response of Schwann cells. Removal of extracellular Ca²⁺ by addition of EGTA to the culture medium suppressed [³H] thymidine incorporation as did the calmodulin inhibitor 48/80. The Ca²⁺ ionophore A23187 increased incorporation. Staurosporin, an inhibitor of protein kinase C (PKC), suppressed [³H] thymidine incorporation while phorbol-12-myristate-13-acetate (PMA) enhanced incorporation. Manipulation of the cAMP system showed that increased cAMP levels inhibited proliferation. Inhibition of protein kinase A by HA 1004 increased the incorporation of [³H] thymidine. Immunostaining for BrdU and glial specific markers together with morphological evaluation of myelin association showed that proliferation occurred in Schwann cells. The results are consistent with a model in which Schwann cell proliferation is enhanced by Ca²⁺ through activation of calmodulin-dependent and/or PKCdependent mechanisms. Inhibition is achieved through the cAMP system. Together, these results show that Schwann cells regulate proliferation differently in an integrated environment, e.g. the nerve structure, than in isolation as primary monocultures. J. Neurosci. Res. 52:530–537, 1998. © 1998 Wiley-Liss, Inc.     

Neurogenesis in the olfactory tubercle and islands of Calleja in the rat

Neurogenesis in the rat olfactory tubercle and islands of Calleja was examined with [³H]thymidine autoradiography. Animals in the prenatal groups were the offspring of pregnant females given an injection of [³H]thymidine on two consecutive gestational days. Ten groups of embryos (E) were exposed to [³H]thymidine on E12–E13, E13–E14 4 E21–E22, respectively. Three groups of postnatal animals (P) were given four consecutive injections of [³H]thymidine on P0–P3, P2–P5, and P4–P7, respectively. On P60, the percentage of labeled cells and the proportion of cells originating during either 24 or 48 h periods were quantified at several anatomical levels. Three populations of neurons were studied:

1. (1) large cells in layer III

2. (2) small to medium-sized cells in layers II and III, and in the striatal bridges

3. (3) granule cells in the islands of Calleja. Neurogenesis is sequential between these three populations with No. 1 oldest and No. 3 youngest. The large neurons in layer III originate mainly between E13 and E16 in a strong lateral-to-medial gradient. Neurons in population No. 2 are generated between E15 and E20, also in a lateral-to-medial gradient; neurogenesis is simultaneous along the superficialdeep plane. Granule cells in the smaller islands of Calleja are generated between E17 and E22 in combined lateral-to-medial and superficial-to-deep gradients. Neurons in the large island of Calleja are generated mainly between E19 and E22 in a strong rostral-to-caudal gradient. Neurogenesis is reduced to < 10% in both populations No. 2 and No. 3, occurring mainly between P0 and P4. The neurogenetic patterns in populations No. 2 and No. 3 are similar to those in the striatum, while neurogenesis in population No. 1 fits into the pattern of the globus pallidus and substantia innominata. These developmental patterns indicate that the olfactory tubercle is a mixed striato-pallidal system rather than an olfactory cortical area. The lateral-to-medial neurogenetic gradient shown in each neuronal population in the olfactory tubercle correlates with both differential anatomical projections and differential neurochemical characteristics along the lateral-medial plane.

Antipsychotics with inverse agonist activity at the dopamine D3 receptor

Summary In NG 108-15 cells expressing the recombinant human D₃ receptor, dopamine agonists enhance [³H]thymidine incorporation and decrease cAMP accumulation. In these cells, but not in wild type cells, haloperidol, fluphenazine, and various other antipsychotics inhibited basal [³H]thymidine incorporation in a concentration-dependent manner. In contrast, other dopamine antagonists such as nafadotride or (+)AJ 76, two D₃-preferring antagonists, were without effect. The concentration-response curve of haloperidol was shifted to the right in presence of nafadotride, with a potency compatible with its nanomolar apparent affinity as neutral antagonist. Pertussis toxin treatment abolished or markedly reduced the responses to haloperidol or fluphenazine. In contrast, no significant enhancement of cAMP accumulation could be observed, under the influence of haloperidol or eticlopride. These data indicate that some dopamine antagonists behave as inverse agonists, and thus appear to inhibit an agonist-independent activity of the D₃ receptor on [³H]thymidine incorporation pathway, but not on the cAMP pathway.     

Histogenesis of the superior colliculus of the albino rat: A tritiated thymidine study

The pattern of generation of neurons in the albino rat superior colliculus has been studied in adult and fetal material. Neurons are generated between embryonic days 12 to 17, with rostrolateral colliculus in advance of caudomedial parts. More of the cells contributing to the deeper layers are generated early, while more of the later generated cells are located superficially. The cells of individual laminae are not formed on specific days as in the cortex, nor are the complicated gradients described previously for the chick optic tectum¹⁸ evident. While the largest cells (found deep in the colliculus) are among the first formed and the small marginal cells among the last, there is in general a broad range of cell size being generated at any one time. The observed patterns are consistent with the concept of simultaneous production of several cell types from the ventricular epithelium on any given day. Studies of material at short times after [³H]thymidine injection allow correlation of the time of arrival of cells in their appropriate layer with time of arrival of afferents. In addition they suggest that factors controlling the final placement of cells in the mature nervous system is a very complex process and may involve some form of intercellular recognition.     

Endogenous opioid systems regulate cell proliferation in the developing rat brain

The role of endogenous opioid systems in modulating the proliferation of developing cerebellar cells was examined autoradiographically in 6-day-old rats. The blockade of endogenous opioid-opioid receptor interaction by naltrexone, a potent opioid antagonist, was accompanied within 1–2 h by an increased proportion of cells incorporating [³H]thymidine. When high doses of naltrexone (50 mg/kg) were administered this index was still elevated 12 h later; however, when low doses of naltrexone (1 mg/kg) were administered the index of labeled cells was decreased markedly. Injection of methionine-enkephalin, an endogenous opioid peptide, also resulted in a decrease in the proportion of cells incorporating [³H]thymidine. Concomitant injection of 1 mg/kg naloxone, however, blocked the inhibitory effects of methionine-enkephalin on cell division but did not itself affect cell generation. These studies demonstrate that endogenous opioid systems can regulate the proliferation of cell populations in the developing nervous system and do so through an inhibitory mechanism.     

Transmitter histochemistry of the rat olfactory bulb. III. Autoradiographic localization of [H]GABA

The distribution of [³H]γ-aminobutyric acid (GABA) labeled elements in rat olfactory bulb was studied by light and electron microscopic autoradiography. [³H]GABA was strongly taken up into glial cells and pericytes in all layers of the bulb. The neuronal uptake of [³H]GABA was mainly seen in certain types of nerve terminals. About one-third of the granule dendritic terminals, some nerve endings of short axon cells, and certain nerve endings of extrabulbar origin showed a strong labeling. Labeling was seen in a small population of the periglomerular, short axon and granule cell bodies. Most cell bodies of these 3 types as well as the mitral cells did not, however, accumulate any appreciable amount of [³H]GABA. The labeling pattern seen after injection of [³H]glycine and [³H]leucine was clearly different from the pattern seen after [³H]GABA injection. The labeling was more uniformly distributed over the components of the neuropil with a considerably higher activity over certain cell somata such as the mitral cells. The present results demonstrate that neuronal uptake and accumulation of [³H]GABA occur into populations of olfactory bulb cells and processes, which from neurophysiological and/or immunohistochemical studies are supposed to use GABA as a neurotransmitter.     

Identification of a saturable uptake system for deoxyribonucleosides at the blood-brain and blood-cerebrospinal fluid barriers

Substances can enter the brain either directly across the blood-brain barrier or indirectly across the choroid plexuses and arachnoid membrane (blood-CSF barrier) into the CSF and then by diffusion into the brain. Earlier studies have demonstrated a saturable thymidine uptake across the blood-CSF barrier, but not across the blood-brain barrier. In this study transport of [³H]thymidine across both barriers was measured in vivo by means of a bilateral vascular brain perfusion technique in the anaesthetised guinea-pig. This method allows simultaneous and quantitative measurement of slowly penetrating solutes into both brain and CSF, under controlled conditions of arterial inflow. The results of the present study carried out over perfusion periods of up to 30 min indicated a progressive uptake of [³H]thymidine into brain and CSF, which was found to be significantly greater than the transport ofd-[¹⁴C]mannitol (a plasma space marker). Furthermore, the addition of 1 mM unlabelled thymidine in the perfusate caused saturation of [³H]thymidine uptake into both brain and CSF. In conclusion, these findings suggest that thymidine can cross both the blood-brain and blood-CSF barriers in the guinea-pig by carrier-mediated transport systems.     

Distribution of [3H]RNA in goldfish optic tectum following intraocular or intracranial injection of [3H]uridine. Evidence of axonal migration of RNA in regenerating optic fibers.

The distribution of [³H]RNA in the goldfish optic tectum following eitherintra-ocular orintracranial injection of [³H]uridine during optic fiber regeneration has been studied by light (LMA) and electron (EMA) microscopic autoradiography.In one group of 4 fish both optic nerves were crushed, and 18 days later [³H]uridine was injected into the right eye. A second group of 5 fish, in which only one optic nerve had been crushed, received intracranial injections of [³H]uridine 18 or 22 days after the crush. All fish were sacrificed 24 days after crushing the optic nerves, a time when regenerating optic fibers have entered the tectum and are establishing functional reconnections. Tecta were fixed in situ with glutaraldehyde, dissected out, and samples were processed for LMA and EMA. Controls were carried out to ensure that [³H]RNA was the only radioactive component present in the tissue after fixation.The distribution of silver grains related to [³H]RNA in intraocularly injected goldfish was different from that following intracranial injection. Following intraocular injection virtually all the [³H]RNA was located in the layers of the left optic tectum (contralateral to the side of intraocular injection) where the regenerating optic fibers course and terminate, whereas virtually no radioactivity was present in the right optic tectum. EMA quantitative analysis of the labeled layers of the left optic tectum revealed that perikarya of cells, most of which are glial cells, had a density of grains related to [³H]RNA of 20–28 g/100 sq.μm; axonal growth cones had a density of 14–24 g/100 sq.μm. Grain densities over non-axonal cell structures were markedly lower, ranging between 3 and 6 g/sq.μm. Grains located over axons and growth cones accounted for 50–60% of all counted grains.Inintracranially injected goldfish, either 2 or 6 days after injection, silver grains were clustered over leptomeninges as well as vessels and parenchymal cells of the tectal strata containing the regenerating optic fibers. In the stratum opticum a high grain density was seen over glial cells, whereas virtually no grains were present over the fascicles of regenerating axons. EMA quantitative analysis revealed a grain density over glial and other parenchymal cells of the stratum opticum of 67 g/100 sq.μm, whereas densities over growth cones and regenerating axons were 1.3 g/100 sq.μm and 1.8 g/100 sq.μm respectively. Grains located over axons and growth cones accounted for 3.3% of all counted grains.On the basis of the present and previous findings it is suggested that followingintraocular injection of [³H]uridine the [³H]RNA present inside regenerating optic axons is transported from the ganglion cells of the retina; on the other hand, the [³H]RNA present in surrounding glial cells is the result of local utilization of [³H]RNA precursors which also migrate from the retina along with the [³H]RNA.It is also concluded that 2 and 6 days followingintracranial injection of [³H]uridine no substantial tranfer of [³H]RNA from glial cells to regenerating optic fibers occurs in the goldfish optic tectum.     

Post- and presynaptic lesions in the CA1 region of hippocampus: Effect on [3H]forskolin and [3H]phorboldibutyrate ester binding

Summary The effect of transient cerebral ischemia and intraventricular injection of kainic acid on adenylate cyclase and protein kinase C as labeled by [³H]forskolin ([³H]FOR) and [³H]phorboldibutyrate ester ([³H]PDBU) in several rat brain microregions was investigated in a quantitative autoradiographic study. Four days after transient four vessel occlusion a 80% loss of [³H]FOR and a 35% loss of [³H]PDBU binding could be measured in the CA1 stratum radiatum of operated Wistar rats as compared to control rats. Four days after intraventricular injection of kainic acid only a marginal loss of [³H]FOR and a 30% increase of [³H]PDBU binding was seen in the CA1 stratum radiatum while in the CA3 stratum lucidum and radiatum respectively a 30% loss of [³H]FOR and no significant change in [³H]PDBU binding was observed. As transient cerebral ischemia and intraventricular kainic acid injection are depleting the hippocampal CA1 region of CA1 pyramidal cells and axons of CA3 pyramidal cells respectively in rat brain, these findings strongly suggest that both adenylate cyclase and protein kinase C are localized in CA1 pyramidal cells of rat hippocampus.Part of this study has been presented at the 16th C.I.N.P. Congress, Munich, August 15–19, 1988.     

Mitotic cycling of radial glial cells of the fetal murine cerebral wall: a combined autoradiographic and immunohistochemical study

Radial glial cells of the embryonic murine cerebral wall are selectively labeled by staining with antibody RC1. In order to study the mitotic cycling of these cells, we combined RC1 immunohistochemistry and autoradiographic analysis following [³H]thymidine injection at 1, 2, 6, 48 h prior to sacrifice. Many radial glial cells, i.e. RC1-positive cells, incorporate the DNA tracer and hence must be mitotically active. Other proliferative cells of the ventricular zone do not stain with RC1. With the transition from S to M phase, the nuclei of the radial glial cells participate in the interkinetic ‘to-and-fro’ nuclear translocation characteristic of the non-radial glial cells of the ventricular zone. The density of radioactive grains over nuclei of both RC1-positive and negative cells of the ventricular zone becomes similarly reduced in the 48 h following the [³H]thymidine incorporation. Thus, the subpopulation of radial glia with nuclei within the ventricular zone which have incorporated the DNA tracer does not appear to become arrested in a prolonged G1 phase. The results suggest that the ventricular zone includes at least two subpopulations of stem cells, neuronal and radial glial. Radial glial cells. i.e. RC1-positive cells, are inferred to serve initially as a progenitor population for new radial glial cells. Later in development, they probably become a source of other cells of astroglial lineage.