Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus.

Knowing the rate of addition of new granule cells to the adult dentate gyrus is critical to understanding the function of adult neurogenesis. Despite the large number of studies of neurogenesis in the adult dentate gyrus, basic questions about the magnitude of this phenomenon have never been addressed. The S-phase marker bromodeoxyuridine (BrdU) has been extensively used in recent studies of adult neurogenesis, but it has been carefully tested only in the embryonic brain. Here, we show that a high dose of BrdU (300 mg/kg) is a specific, quantitative, and nontoxic marker of dividing cells in the adult rat dentate gyrus, whereas lower doses label only a fraction of the S-phase cells. By using this high dose of BrdU along with a second S-phase marker, [(3)H]thymidine, we found that young adult rats have 9,400 dividing cells proliferating with a cell cycle time of 25 hours, which would generate 9,000 new cells each day, or more than 250,000 per month. Within 5-12 days of BrdU injection, a substantial pool of immature granule neurons, 50% of all BrdU-labeled cells in the dentate gyrus, could be identified with neuron-specific antibodies TuJ1 and TUC-4. This number of new granule neurons generated each month is 6% of the total size of the granule cell population and 30-60% of the size of the afferent and efferent populations (West et al. [1991] Anat Rec 231:482-497; Mulders et al. [1997] J Comp Neurol 385:83-94). The large number of the adult-generated granule cells supports the idea that these new neurons play an important role in hippocampal function.

Restoring production of hippocampal neurons in old age.

The production of hippocampal granule neurons continues throughout adulthood but dramatically decreases in old age. Here we show that reducing corticosteroid levels in aged rats restored the rate of cell proliferation, resulting in increased numbers of new granule neurons. This result indicates that the neuronal precursor population in the dentate gyrus remains stable into old age, but that neurogenesis is normally slowed by high levels of corticosteroids. The findings further suggest that decreased neurogenesis may contribute to age-related memory deficits associated with high corticosteroids, and that these deficits may be reversible.

Stem cells and neurogenesis in the adult brain.

A new interest in replacing neurons lost to trauma or disease has been generated by findings that challenge the traditional view of the static (irreparable) adult brain. The discovery of stem cells and neurogenesis in the adult central nervous system is responsible for much of this interest.

Regulation of neurogenesis by growth factors and neurotransmitters.

The generation of neurons and glia in the developing nervous system is likely to be regulated by extrinsic factors, including growth factors and neurotransmitters. Evidence from in vivo and/or in vitro systems indicates that basic fibroblast growth factor, transforming growth factor (TGF)-alpha, insulin-like growth factor-1, and the monoamine neurotransmitters act to increase proliferation of neural precursors. Conversely, glutamate, gamma-aminobutyric acid, and opioid peptides are likely to play a role in down-regulating proliferation in the developing nervous system. Several other factors, including the neuropeptides vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide, as well as the growth factors platelet-derived growth factor, ciliary neurotrophic factor, and members of the TGF-beta family, have different effects on proliferation and differentiation depending on the system examined. Expression of many of these factors and their receptors in germinal regions of the central nervous system suggests that they can act directly on precursor populations to control their proliferation. Together, the findings discussed here indicate that proliferation and cell fate determination in the developing brain are regulated extrinsically by complex interactions between a relatively large number of growth factors and neurotransmitters.

Adrenal steroids and N-methyl-D-aspartate receptor activation regulate neurogenesis in the dentate gyrus of adult rats through a common pathway.

Adrenal steroids and N-methyl-D-aspartate receptor activation have both been shown to regulate the rate of proliferation of granule neuron progenitor cells in the dentate gyrus of adult rats [Cameron H. A. and Gould E. (1994) Neuroscience 61, 203-209; Cameron H. A. et al. (1995) J. Neurosci. 15, 46874692]. Parallels between the actions of these two factors suggest that they may regulate cell division through a common pathway. This hypothesis was tested by altering both of the factors simultaneously and determining whether the effects were additive. The results of this study demonstrate that alterations in N-methyl-D-aspartate receptor activation block the effects of corticosterone level on cell proliferation; N-methyl-D-aspartate blocks the adrenalectomy-induced increase in [3H]thymidine-labelled cell density in the dentate gyrus, whereas the N-methyl-D-aspartate receptor antagonist dizocilpine maleate (MK-801) prevents the corticosterone-induced decrease in proliferating cells. This finding suggests that adrenal steroids and N-methyl-D-aspartate receptor activation regulate granule cell production in the adult rat dentate gyrus through a common pathway and that N-methyl-D-aspartate receptor activation operates downstream of corticosterone in this pathway.

Adrenal steroids suppress granule cell death in the developing dentate gyrus through an NMDA receptor-dependent mechanism.

Treatment with the NMDA receptor antagonist MK-801 prevented the adrenal steroid-induced suppression of cell death, determined by both morphological identification of pyknotic cells and TUNEL staining, in the dentate gyrus in rat pups. This finding suggests that adrenal steroids naturally promote granule cell survival via NMDA receptor activation.

Early NMDA receptor blockade impairs defensive behavior and increases cell proliferation in the dentate gyrus of developing rats.

These studies were conducted to determine whether (a) early N-methyl-D-aspartate (NMDA) receptor blockade impairs defensive behavior and (b) a relationship exists between defensive behavior and the production of granule cells in the dentate gyrus. Rat pups were treated with different doses of the NMDA receptor antagonist CGP 43487 on postnatal day (P) 5, and their behavior was observed following exposure to an unfamiliar adult male rat, a potential predator, on P13, P20, and P30. A dose-dependent impairment in freezing behavior was observed in rat pups treated with NMDA receptor antagonist on P13, P20, but not P30. Moreover, a dose-dependent increase in the number of (3)H-thymidine-labeled cells in the dentate gyrus was detected following CGP 43487 treatment, suggesting that an inverse relationship exists between cell proliferation and freezing behavior in rat pups following NMDA receptor blockade.

Distinct populations of cells in the adult dentate gyrus undergo mitosis or apoptosis in response to adrenalectomy.

Granule neurons of the rat dentate gyrus are born in adulthood as well as during development. Apoptotic cell death also occurs normally in this population throughout the life of the rat. Removal of adrenal steroids results in both increased production and increased degeneration of dentate gyrus granule cells. In order to determine whether the age of a cell affects its response to adrenalectomy (ADX), the numbers of dentate gyrus cells of different ages were assessed following ADX or sham operation. Older cells, i.e., those labeled with the thymidine analog bromodeoxyuridine (BrdU) on postnatal day (P) 6, were reduced in number following ADX on P60, and some had the morphologic characteristics of degenerating cells, indicating that significant numbers of mature cells die in response to ADX. In contrast, the number of younger cells, labeled with 3H-thymidine or BrdU in adulthood, 24 hours or 2 weeks before ADX, did not decrease, suggesting that these less mature cells do not die following ADX. An increase in the number of cells that are immunoreactive for proliferating cell nuclear antigen, a marker of dividing or recently mitotic cells, indicates that immature dentate gyrus cells divide following ADX. These results suggest that following ADX, mature cells born during the 1st postnatal week die, whereas immature cells divide.

Regulation of neuronal birth, migration and death in the rat dentate gyrus.

The granule cell population of the rat dentate gyrus forms over an extended period which begins during gestation and continues into adulthood. During the embryonic period, the postnatal period and in adulthood, granule cells proliferate, migrate and degenerate. We have found that granule cell production is dependent on the levels of circulating adrenal steroids and NMDA receptor-mediated excitatory input throughout life. In general, increases in adrenal steroid levels or NMDA receptor activation diminish the rate of cell proliferation whereas decreases in adrenal steroid levels or NMDA receptor activation increase the rate of cell production. This paper describes the regulation of granule cell proliferation, migration and survival by adrenal steroids and excitatory input and presents evidence that these factors may affect dentate gyrus-mediated behaviors.

Regulation of adult neurogenesis by excitatory input and NMDA receptor activation in the dentate gyrus.

The effects of afferent input and N-methyl-D-aspartate (NMDA) receptor activation on neurogenesis were examined in an intact system, the rat dentate gyrus, where neurons are naturally born in the adult. In the adult dentate gyrus, activation of NMDA receptors rapidly decreased the number of cells synthesizing DNA, whereas blockade of NMDA receptors rapidly increased the number of cells in the S phase identified with 3H-thymidine. Acute treatment with NMDA receptor antagonists increased the birth of neurons and increased the overall density of neurons in the granule cell layer. Lesion of the entorhinal cortex, the main excitatory afferent population to the granule neurons, also increased the birth of cells in the dentate gyrus. These results suggest that adult neurogenesis in the dentate gyrus of the rat is altered by afferent input, via NMDA receptors, and may be regulated naturally by endogenous excitatory amino acids.

Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus.

The dentate gyrus of the rat produces new granule neurons well into adulthood. In the adult, newly born granule neurons migrate from the hilus to the granule cell layer, receive synaptic input, extend axons into the mossy fiber pathway, and express a neuronal marker. No previous studies have identified factors that regulate neuronal birth in the adult dentate gyrus. In order to determine whether glucocorticoids control neurogenesis in the adult dentate gyrus, the effects of adrenal steroid manipulations on neuronal birth were assessed using [3H]thymidine autoradiography and immunohistochemistry for the neuronal marker neuron specific enolase. Acute treatment with corticosterone produced a significant decrease in the density of [3H]thymidine-labeled cells in the hilus of the dentate gyrus. In contrast, removal of endogenous adrenal steroids stimulated increased neuronal birth; adrenalectomy resulted in a significant increase in the number of neuron specific enolase-immunoreactive [3H]thymidine labeled cells in the granule cell layer compared to sham operation. Replacement of corticosterone to adrenalectomized rats after [3H]thymidine injection did not substantially alter the increase in neurogenesis observed following adrenalectomy, even though this replacement protects cells from adrenalectomy-induced cell death. These results indicate that the rate of neurogenesis in the dentate gyrus of the adult rat is dependent upon the levels of circulating adrenal steroids.

Blockade of NMDA receptors increases cell death and birth in the developing rat dentate gyrus.

Excitatory input regulates cell birth and survival in many systems. The granule cell population of the rat dentate gyrus is formed primarily during the postnatal period. Excitatory afferents enter the dentate gyrus and begin to form synapses with granule cells during the first postnatal week, the time of maximal cell birth and death. In order to determine whether excitatory input plays a role in the regulation of cell birth and survival in the developing granule cell layers and their germinal regions, the subependymal layer and hilus, we treated rat pups with the N-methyl D-aspartate (NMDA) receptor antagonists MK-801, CGP 37849, or CGP 43487 during the first postnatal week and examined the numbers of 3H-thymidine-labeled cells, pyknotic cells, and healthy cells in these regions. In order to determine the cell type that was affected, sections from brains of MK-801-treated rats were processed for 3H-thymidine autoradiography combined with immunohistochemistry for the marker of radial glia, vimentin, and the marker of mature astrocytes, glial fibrillary acidic protein (GFAP). Within the dentate gyrus, NMDA receptor blockade resulted in the following changes: (1) the density of 3H-thymidine-labeled cells was increased, (2) the density of pyknotic cells was increased, (3) the density of 3H-thymidine-labeled pyknotic cells was increased, and (4) the density of healthy cells was decreased. The infrapyramidal blade/hilus showed changes throughout its extent, whereas the suprapyramidal blade showed changes only at the rostral level. No change in the numbers of 3H-thymidine-labeled vimentin-immunoreactive or GFAP-immunoreactive cells was observed in the dentate gyrus with MK-801 treatment, indicating that glia are not primarily affected by NMDA receptor blockade. Blockade of NMDA receptors resulted in gross morphologic changes in the dentate gyrus; in most cases, the infrapyramidal blade was indistinguishable from the hilus. Moreover, in several brains of animals treated with CGP 37849 or CGP 43487 on postnatal day (P)5, an abnormal aggregation of cells was observed ventral to the normal location of the infrapyramidal blade. This cellular cluster contained many pyknotic and 3H-thymidine-labeled cells and may represent cells that normally comprise the infrapyramidal blade. Dramatic changes to the subependymal layer were also seen following NMDA receptor blockade. The cross-sectional area of this region was significantly increased with MK-801, CGP 37849, or CGP 43487 treatment and contained a high density of 3H-thymidine-labeled cells and 3H-thymidine-labeled pyknotic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat.

In order to determine whether newly born cells in the dentate gyrus of the adult rat express the neuronal marker, neuron-specific enolase, or the glial marker, glial fibrillary acidic protein, we performed combined immunohistochemistry and autoradiography on brains from adult rats perfused at various times ranging from 1 h to four weeks following [3H]thymidine administration. Light-microscopic examination revealed a negligible number of [3H]thymidine-labeled cells showing neuron-specific enolase immunoreactivity during mitosis. However, by two weeks after [3H]thymidine administration, a significant increase in the density of [3H]thymidine-labeled neuron-specific enolase-immunoreactive cells was detected. Three weeks following [3H]thymidine injection the majority of [3H]thymidine-labeled cells (> 70%) were immunoreactive for the neuronal marker. At the four-week time-point, [3H]thymidine-labeled neuron-specific enolase-immunoreactive cells were indistinguishable from neighboring granule cells. In contrast, glial fibrillary acidic protein immunoreactivity was observed in a small but significant number of [3H]thymidine cells at the 1-h time-point and the proportion of labeled cells that were immunoreactive for this cell marker did not increase with time. [3H]Thymidine-labeled cells that were immunoreactive for glial fibrillary acidic protein typically showed morphologic characteristics of radial glia at all time-points. At the 1-h time-point, the majority of [3H]thymidine-labeled cells were observed in the hilus (> 60%) with the remainder being located in the granule cell layer. However, with a four-week survival-time most [3H]thymidine-labeled cells (> 85%) were located in the granule cell layer. The majority of newly born cells in the adult dentate gyrus differentiate into neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal steroid receptor immunoreactivity in cells born in the adult rat dentate gyrus.

Several lines of evidence indicate that cell birth in the adult rat dentate gyrus is regulated by adrenal steroids. The expression of adrenal steroid receptors by mitotic cells in the dentate gyrus would support the hypothesis that these hormones act directly on granule cell progenitors. We performed a survival time course of in vivo [3H]thymidine autoradiography combined with immunohistochemistry for mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) and found that very few [3H]thymidine labeled mitotic cells express these receptors. By 4 weeks following [3H]thymidine administration, the vast majority of [3H]thymidine labeled cells were immunoreactive for MR and GR. These results suggest that adrenal steroids do not act directly on granule cell progenitors in the adult rat dentate gyrus.

Phenytoin prevents stress- and corticosterone-induced atrophy of CA3 pyramidal neurons.

Repeated daily restraint stress and daily corticosterone administration to adult male Sprague-Dawley rats leads to decreases in the number of branch points and length of dendrites of CA3 pyramidal neurons of the hippocampal formation. This decrease is prevented by daily administration of the antiepileptic drug phenytoin (Dilantin), which is known to interfere with excitatory amino acid release and actions. Phenytoin had no obvious effect on behavior during and after stress and failed to prevent stress-induced reduction of body weight gain and stress-induced increases of adrenal weight relative to body weight; it also failed to attenuate glucocorticoid-induced diminution of the size of the thymus gland, indicating that it does not directly antagonize glucocorticoid actions. Stress- and corticosterone-induced effects on dendritic length and branch point number are more pronounced on the apical, as opposed to the basal, CA3 dendrites that receive the largest mossy fiber input from the dentate gyrus. Because phenytoin is also known to prevent ischemic damage, these results are consistent with a model in which stress- and corticosterone-induced CA3 dendritic atrophy is produced by excitatory amino acids released from the mossy fibers.

Adrenal hormones suppress cell division in the adult rat dentate gyrus.

The rat dentate gyrus is unusual among mammalian brain regions in that it shows cell birth well into adulthood. During development, dentate gyrus cell birth is regulated by adrenal steroids. However, it is presently unknown whether cell division in the adult is also mediated by these same factors. In order to determine whether this is the case, we combined adrenalectomy, with or without corticosterone (CORT) replacement, and 3H-thymidine autoradiography, Nissl staining, and immunohistochemistry for the glial cell markers vimentin and glial fibrillary acidic protein (GFAP) as well as for the neuronal marker neuron-specific enolase. Removal of circulating adrenal steroids resulted in a greater density of both GFAP-immunoreactive and vimentin-immunoreactive cells compared to sham-operated animals; CORT replacement prevented increases in both of these cell types. The increase in the density of vimentin-immunoreactive cells probably resulted from an increase in the birth of these cells, as adrenalectomized rats showed greater numbers of 3H-thymidine-labeled vimentin-positive cells compared to sham rats. In contrast, no changes in the number of 3H-thymidine-labeled GFAP-positive cells were observed with adrenalectomy, indicating that the increase in this cell type probably does not involve cell birth. In addition, the density of 3H-thymidine-labeled cells that were not immunoreactive for either glial cell marker and that showed neuronal characteristics was dramatically increased with adrenalectomy. These results suggest that adrenal hormones normally suppress the birth of both glia and neurons in the adult rat dentate gyrus.

Investigation of selected patients with hypertension by the rapid-sequence intravenous urogram.

The rapid-sequence intravenous urogram (IVU) has tended to fall from favour for investigating hypertension because of its perceived imprecision for detecting renovascular disease. However, no study has examined the value of the IVU as a screening test in appropriately selected patients. We have analysed the diagnostic yield of the rapid-sequence IVU in hypertensive patients selected for features suggesting renal or renovascular disease in a retrospective review of case records from a hypertension clinic. The IVU was abnormal in 27% (95% CI 21-32%) of 241 consecutive patients. The most common abnormalities were chronic pyelonephritis (6%); proven renovascular disease (5%); stone (4%); possible renovascular disease and simple cyst (each 3%); hydronephrosis (2%); and tumour and active tuberculosis (each 1%). The IVU led to intervention aiming to correct hypertension in 5% (95% CI 2-8%) of patients, and revealed an abnormality needing intervention in its own right in 4% (95% CI 2-6%). The IVU led to unnecessary invasive investigation in 3% of cases. Individual abnormalities could not be predicted from the clinical or laboratory features. The initial investigation in hypertensive patients with suspected renal or renovascular disease should be a general purpose test able to detect a wide range of abnormalities. The rapid-sequence IVU is the only single test capable of satisfying this requirement. In patients with features suggesting renovascular disease, a normal rapid-sequence IVU excludes renovascular disease with 93% probability, but is an imperfect screening test since it fails to diagnose about 20% of cases. Renal arteriography should be done despite a normal IVU when it is essential to exclude renovascular disease.

Expression of adrenal steroid receptors by newly born cells and pyknotic cells in the dentate gyrus of the postnatal rat.

Neurogenesis and cell survival in the postnatal rat dentate gyrus are regulated by adrenal steroids. Increases in the circulating levels of glucocorticoids and mineralocorticoids result in decreased cell birth and increased cell survival in the dentate gyrus during the first postnatal week. It is presently unknown whether the effects of these hormones on cell birth and survival are direct or indirect. The expression of adrenal steroid receptors by dentate gyrus neuroblasts and pyknotic cells would support the contention that adrenal steroids directly affect neurogenesis and cell death. To determine whether or not this is the case, immunohistochemistry for the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) was combined with short-survival [(3)H]thymidine autoradiography in rat pups during the first postnatal week. Light microscopic analysis revealed immunoreactivity for both GRs and MRs in the dentate gyrus. Approximately 50% of all pyknotic cells showed GR-like immunoreactivity, whereas no examples of MR-immunoreactive pyknotic cells were detected. In addition, some [(3)H]thymidine-labeled cells were immunoreactive for GRs (approximately 10%) but no [(3)H]thymidine-labeled MR-immunoreactive cells were observed. The relatively high percentage of pyknotic cells that were GR-immunoreactive suggests that adrenal steroids influence cell survival directly through GRs. In contrast, the relatively low percentage of [(3)H]thymidine-labeled cells expressing GRs indicates that adrenal steroids influence cell birth indirectly through an as yet unidentified mechanism.

Adrenal steroids regulate postnatal development of the rat dentate gyrus: II. Effects of glucocorticoids and mineralocorticoids on cell birth.

Unlike the majority of mammalian brain regions, the rat dentate gyrus undergoes maximal cell birth and cell death during the same developmental time period. Granule cell birth and death peak at the end of the first postnatal week. We have found that manipulations of glucocorticoid levels during the stress hyporesponsive period profoundly influence the density of pyknotic cells in the dentate gyrus while apparently not affecting the density of healthy cells. This raises the possibility that glucocorticoids are regulating processes in addition to cell death, i.e., cell birth. In order to determine whether increases in circulating glucocorticoids or mineralocorticoids affect the birth of cells in the developing dentate gyrus, 3H-thymidine autoradiography was performed on brains of rat pups treated with either corticosterone or aldosterone during the first postnatal week. Quantitative analysis of 3H-thymidine-labelled cells revealed significant decreases in the density of labelled cells in the granule cell layers with both corticosterone and aldosterone treatment. In these same brains, significant decreases in the density of pyknotic cells were also observed in the granule cell layers. However, no changes in the numbers of 3H-thymidine-labelled pyknotic cells were observed with any treatment. Increases in circulating corticosterone or aldosterone resulted in significant increases in the density of both 3H-thymidine-labelled and pyknotic cells in the hilus. These results suggest that dentate gyrus cell birth and cell death are related and that these processes are regulated by adrenal steroids.

Organization of mitochondria in olfactory bulb granule cell dendritic spines.

In contrast to dendritic spines with only postsynaptic functions, the spines of olfactory bulb granule cells subserve both pre- and postsynaptic roles. In single sections these spines were previously seen to contain mitochondria, most likely needed to provide energy for presynaptic functions, but their frequency and distribution were unknown. In order to understand the organization of mitochondria in these specialized dendritic appendages, we have studied the geometry and cytoplasmic organization of granule cell spines with computer-assisted reconstructions of serial electron micrographs. The spine heads were seen to be elliptical in shape with a single pair of reciprocal synapses on the concave face apposed to the mitral/tufted cell dendrite. Mitochondria were found localized in the spine neck as well as the spine head and often extended between the two compartments. Based on their variable distribution it seems reasonable to suggest that these mitochondria are motile and move in and out of spine compartments from the parent dendrite. Spine apparatus was apparent in most of the spines as membrane bound cisterns of smooth endoplasmic reticulum located close to mitochondria. The possible role of spine apparatus in facilitating the movement of mitochondria in the necks and heads of granule cell spines in the absence of microtubules is discussed.