Implication of corticosteroid receptors in the regulation of hippocampal structural plasticity

The dentate gyrus is one of the few areas of the adult brain that continues to produce neurons and to express the embryonic polysialylated isoforms of neuronal cell adhesion molecules (PSA-NCAM). The stress hormone corticosterone exerts a complex modulation on neurogenesis and PSA-NCAM, and previous studies have shown that mature granule cells require corticosterone for their survival. Thus, the aim of our work was to investigate the respective role of the different corticosteroid receptors on these three parameters in adrenalectomized rats. It was found that treatment with a low dose of the mineralocorticoid receptor agonist, aldosterone, prevents only the adrenalectomy-induced increase in cell death. Treatment with a higher dose of aldosterone normalized cell proliferation whereas PSA-NCAM expression was normalized only by treatment with the glucocorticoid receptor agonist, RU 28362. It is concluded that stimulation of the mineralocorticoid receptor is sufficient to mediate the effects of corticosterone on neurogenesis and to protect mature cells from cell death whereas stimulation of the glucocorticoid receptor is necessary to modulate PSA-NCAM expression.     

Effects of adrenal steroid manipulations and repeated restraint stress on dynorphin mRNA levels and excitatory amino acid receptor binding in hippocampus

Adrenal steroid and stress effects were determined in hippocampus on levels of dynorphin (DYN) mRNA, expressed in dentate gyrus, and excitatory amino acid receptors, measured in Ammon's horn and dentate gyrus. Adrenalectomy (ADX) decreased DYN mRNA levels in dentate gyrus and replacement with aldosterone (ALDO), a specific type I adrenal steroid receptor agonist, prevented the decrease. Ru28362, a specific type II receptor agonist, had no effect. Likewise, kainate receptor binding to the stratum lucidum and hilus region of dorsal hippocampus was decreased after ADX and this decrease was prevented by ALDO but not by Ru28362 treatment. Similar though smaller effects were found for CNQX binding to AMPA receptors but only in the dentate gyrus molecular or infra- and supragranular layers. Although corticosterone (CORT) treatment of intact rats (40 mg/kg for 3 weeks) elevated DYN mRNA levels in dentate gyrus, up to 14 days of daily restraint stress (1 or 6 h/day) had no significant effect. Neither CORT treatment nor repeated restraint stress altered NMDA and non-NMDA glutamate receptors in hippocampus. The results of this study showing ADX-induced decreases of DYN mRNA and CNQX binding in dentate gyrus and decreased kainate binding in mossy fiber terminal regions are consistent with morphological evidence showing that adrenal steroids maintain normal integrity and structure of dentate gyrus neurons and do so via type I adrenal steroid receptors. These same parameters are apparently not sensitive to chronic restraint stress although the effects of other stressors must be examined.     

Role of adrenal steroid mineralocorticoid and glucocorticoid receptors in long-term potentiation in the CA1 field of hippocampal slices

We previously demonstrated in the dentate gyrus (DG) of anesthetized and freely behaving rats that both acute as well as chronic administration of corticosterone produces a suppression in long-term potentiation (LTP). In subsequent studies we showed, again in the DG, that activation of the two types of adrenal steroid receptors (mineralocorticoid (MR) and glucocorticoid (GR)) produce biphasic effects on synaptic plasticity; activation of MR produces an enhancement while activation of GR produces a suppression in LTP. In a separate study, we further demonstrated in rats administered the specific GR agonist RU 28362 that high-frequency stimulation, which normally produces LTP, instead produced long-term depression (LTD) in these animals. In the present study we investigated the effects of MR and GR activation by adrenal steroids on synaptic plasticity of the hippocampal CA1 field, but we studied this ex vivo, in a slice preparation. The results indicate that, as in our studies in the DG, adrenal steroids produce biphasic effects: in ADX rats, aldosterone (a specific MR agonist) enhanced while RU 28362 suppressed synaptic plasticity. Unlike the in vivo preparation, however, rarely was LTD observed in the animals receiving RU 28362. Also, ADX itself did not produce noticeable effects on synaptic plasticity. The present results are in agreement with previous studies showing that elevations in corticosterone or an acute episode of experimentally induced stress in vivo causes a suppression in LTP in the hippocampal CA1 field, in vitro.     

Effects of mineralocorticoid and glucocorticoid receptors on long-term potentiation in the CA3 hippocampal field

We have previously shown that the two types of adrenal steroid receptors, mineralocorticoid MR. and glucocorticoid GR. produce opposite effects on long-term potentiation LTP. in the dentate gyrus in vivo. and CA1 hippocampal field in vitro. More specifically, MR activation enhanced and prolonged LTP, whereas GR activation suppressed LTP in these areas and also produced a long-term depression LTD. of the synaptic response. In the present experiment we investigated acute effects of MR and GR activation on LTP induction in the mossy fiber and commissural associational input to the CA3 hippocampal field, since the mechanisms underlying LTP induction in these two pathways differ, the former being N-methyl-D-aspartate receptor NMDAR. independent while the latter being NMDAR-dependent. Rats were either adrenalectomized ADX or adrenally intact. ADX animals were acutely injected with either the specific MR agonist, aldosterone, the specific GR agonist RU 28362 or vehicle. One hour following the injection, the animals were prepared for electrophysiological recording stimulation. Field potential recordings were performed in the radiatum or laconosum moleculare layers of the CA3 field, with stimulation of either the mossy fibers or the commissural associational input from the contralateral hemisphere. We also replicated our previous findings by recording in the dentate gyrus with stimulation of the medial perforant pathway, in the same animals. As observed in our previous study in the dentate gyrus, we found an enhancement and a suppression of LTP with MR and GR activation, respectively. Similarly, for the commissural associational input to CA3, MR activation enhanced LTP, while GR activation reduced it. In contrast, for the mossy fiber input to CA3, neither MR nor GR activation significantly affected LTP induction. These results indicate that adrenal steroids may modulate LTP induction in the hippocampus via an interaction with glutamatergic NMDAR.     

Stress inhibits the proliferation of granule cell precursors in the developing dentate gyrus

The granule cell population of the dentate gyrus is produced predominantly during the postnatal period in rats. Previous studies have shown that experimental increases in the levels of adrenal steroids suppress the proliferation of granule cell precursors during the first postnatal week, the time of maximal neurogenesis in the dentate gyrus. These findings raise the possibility that stressful experiences that elevate adrenal steroid levels may inhibit the production of granule neurons, and thus alter the development of the dentate gyrus. To test this possibility, we exposed naive rat pups to the odors of a known predator, adult male rats, and examined both plasma corticosterone levels and the number of [3]H-thymidine labeled cells in the dentate gyrus. A single exposure of rat pups to adult male rat odor elevated corticosterone levels immediately and diminished the number of [3]H-thymidine labeled cells in the granule cell layer by 24 h later. These results suggest that stressful experiences suppress the production of granule neurons in the developing dentate gyrus.     

Quantification of type I and II adrenal steroid receptors in neuronal, lymphoid and pituitary tissues

Circulating lymphocytes are frequently used to study glucocorticoid receptor (GR) regulation in various clinical disease states, such as depression. Since little is known about the relationship between lymphoid and neuronal GR, type II adrenal steroid receptors (i.e., GR) were quantitated in neuronal (hippocampus, frontal cortex, hypothalamus), lymphoid (circulating lymphocytes, spleen, thymus) as well as pituitary tissues of adrenal-intact and 1 day adrenalectomized (ADX) rats using the selective type II receptor ligand, [3H]RU 28362. Specific, high affinity (dissociation constant = 0.2-0.3 nM) type II receptors were present in all tissues examined with the density in 1 day ADX rats being thymus greater than frontal cortex = spleen greater than hippocampus = pituitary greater than hypothalamus greater than lymphocytes. Adrenal intact rats had fewer type II receptors in frontal cortex, hippocampus and spleen as compared to 1 day ADX rats. Dose-response competition studies using [3H]RU 28362 and various unlabelled steroids revealed a binding profile indicative of a type II receptor with the potency being RU 28362 greater than triamcinolone acetonide greater than dexamethasone = corticosterone much greater than aldosterone in both whole brain and spleen soluble fractions. In contrast to the high concentration of type II receptors in the various tissues, the density of type I (i.e., mineralocorticoid) receptors was very low or nondetectable in the same tissues of 1 day ADX rats with the notable exception of the hippocampus where there were approximately comparable levels of both receptors. These results document the widespread distribution of type II adrenal steroid receptors in neuronal and lymphoid tissues which are similar in affinity and steroid specificity.     

Adrenal steroid regulation of neuropeptide Y (NPY) mRNA: differences between dentate hilus and locus coeruleus and arcuate nucleus

Regulation by adrenal steroids of neuropeptide Y (NPY) mRNA was investigated in hilus of the dentate gyrus, arcuate nucleus of hypothalamus and locus coeruleus (LC) of the adult rat brain. Adrenalectomy (ADX) increased NPY mRNA in hilus but decreased NPY mRNA levels in arcuate nucleus and LC. Using a steroid replacement paradigm previously shown to discriminate between Type I and Type II adrenal steroid receptor mediated effects, it was shown that Type I receptor stimulation by aldosterone prevented the ADX-induced increase of NPY mRNA in hilus, whereas Type II receptor stimulation by Ru28362 prevented the ADX-induced decrease in NPY mRNA in arcuate nucleus and LC. The results for hilus are consistent with evidence for a role of Type I receptors in maintaining levels of a number of gene products associated with neurotransmission. The different regulation in hilus from that in arcuate and LC indicate, along with evidence for regulation of NPY expression by insulin, NGF and cyclic AMP and phorbol esters, that the adrenal steroid regulation of NPY gene expression is part of a complex set of regulatory mechanisms that depend on the brain region and cell type.     

Gluco- and mineralocorticoid receptor-mediated regulation of neurotrophic factor gene expression in the dorsal hippocampus and the neocortex of the rat

Gluco- and mineralocorticoid receptors (GR and MR) act via common promoter elements but may exert different effects on gene regulation in various regions of the forebrain. In order to separately analyse the role of GR and MR in the regulation of neurotrophic factor genes and their receptors, we used adrenalectomy and subsequent hormone injections in the rat as a model system. Twenty-four hours after adrenalectomy rats were injected with a single dose of corticosterone (2 and 10 mg/kg), aldosterone (0.5 mg/kg) or the synthetic glucocorticoid agonist RU 28362 (4 mg/kg). Gene expression of basic fibroblast growth factor (bFGF) and its high-affinity receptors [fibroblast growth factor receptor subtypes 1-3 (FGF-R1, FGF-R2, FGF-R3)], as well as brain-derived growth factor (BDNF) and neurotrophin-3 (NT-3) was analysed at 4 h after the hormone injection in CA1-CA4 (cornus of Ammon areas of the hippocampus) and dentate gyrus of the dorsal hippocampus and in neocortex by means of in situ hybridization. We found that bFGF is regulated in CA2, CA3 and dentate gyrus by GR and MR together, and in CA1, CA4 and neocortex by GR alone. FGF-R2 expression in the hippocampus seems to be regulated only by MR, while BDNF expression appears to depend on both receptors. FGF-R1, FGF-R3 and NT-3 were only moderately affected by the hormone activation of GR and MR acting in concert or alone in the various regions. Thus, the present findings suggest that the adrenal cortical system through GR and MR participate in the control of neurotrophic factor signalling in a highly subregion- and cellular-dependent manner.     

Adrenal steroids regulate postnatal development of the rat dentate gyrus: I. Effects of glucocorticoids on cell death.

The rat dentate gyrus undergoes a period of naturally occurring cell death during the first postnatal week. In the adult rat, removal of circulating adrenal steroids by adrenalectomy is followed by massive death in the granule cell layer, thus raising the possibility that developmental cell death results from low levels of these hormones. Interestingly, the first two postnatal weeks of life in the rat, termed the stress hyporesponsive period, are characterized by very low levels of adrenal steroids. In order to determine whether low levels of adrenal steroids enable developmental cell death to occur in the dentate gyrus, we examined the density of pyknotic and healthy cells in the dentate gyrus of rat pups which received one of the following treatments: (1) injections of the endogenous rat glucocorticoid corticosterone during the first postnatal week, or (2) adrenalectomy at the time when glucocorticoid levels normally rise. Quantitative analysis of the density of pyknotic cells in the granule cell layers revealed significant decreases with corticosterone treatment by the end of the first postnatal week. In these same brains, treatment with corticosterone resulted in a substantial increase in the density of pyknotic cells in the hilus. Adrenalectomy resulted in a significant increase in the density of pyknotic cells in the granule cell layer as well as in the hilus. Despite the dramatic alterations in the density of pyknotic cells with both increases and decreases in glucocorticoid levels, the density of healthy cells remained the same. These observations suggest that glucocorticoids regulate several processes, possibly including neurogenesis and migration, in addition to cell death.     

Long-Term Adrenalectomy can Decrease or Increase Hippocampal Dentate Gyrus Volumes

Male and female Long-Evans adult rats were adrenalectomized and sacrificed 6 weeks later to determine whether dentate gyrus damage would differ in females and males. A subset of adrenalectomized rats of both sexes had significantly reduced dentate gyrus volumes compared to the same sex SHAM operated rats. The remainder of the male and female adrenalectomized rats which did not have clear dentate gyrus damage had significantly larger dentate gyrus volumes compared to the same sex SHAM rats. The dentate gyrus volumes of all adrenalectomized rats were significantly correlated with two indices of residual hormonal levels (Na⁺/K⁺ ratios and body weight gain 6 weeks after surgery), indicating that endogenous corticosterone levels may be a determining factor in the response of the dentate gyrus to adrenalectomy. These dentate gyrus volumetric changes could not be attributed to tissue shrinkage as there were no changes in CA3 volumes in any of the groups. These results suggest that long-term adrenalectomy can result in either increased or decreased dentate gyrus volumes and that the adrenal steroid levels of each individual adrenalectomized rat may be the factor determining the direction of the dentate gyrus volumetric response.     

Dexamethasone enhances serum deprivation-induced necrotic death of rat C6 glioma cells through activation of glucocorticoid receptors

Glucocorticoids have been shown to be neurotoxic and appear to play a role in neuronal cell loss during aging and following neuropathological insults. However, very little is known about the effects of these steroid hormones on glial cells. The effect of the synthetic glucocorticoid dexamethasone (DEX) on glial cell viability was therefore examined by measuring neutral red uptake into rat C6 glioma cells. Serum deprivation markedly reduced cell viability, and this effect was significantly enhanced by DEX. Electrophoretic analysis showed that the cell damage induced by either serum deprivation alone or in combination with DEX was not accompanied by the degradation of DNA into nucleosomic fragments. Electron microscopic studies confirmed that serum deprivation and glucocorticoid treatment caused necrotic cell death. Furthermore, the effect of DEX on cell viability could be mimicked by the glucocorticoid receptor agonist RU28362, and completely prevented by the glucocorticoid receptor antagonist RU38486. These results indicate that dexamethasone can enhance the necrotic death of glioma cells induced by serum deprivation, suggesting that glucocorticoids may be involved in the chronic alteration of brain function arising from neuropathological damage to glial cells.     

Glucocorticoid regulation of glial responses during hippocampal neurodegeneration and regeneration

Glucocorticoids can prevent or accelerate neurodegeneration in the adult rat hippocampus. To investigate these actions of glucocorticoids, we previously cloned genes from the hippocampus. Adrenalectomy specifically increased glial fibrillary acidic protein and transforming growth factor (TGF)-β1 mRNAs in the dentate gyrus and these effects were dependent on induced apoptosis. Corticosterone treatment prevented apoptosis, and decreased glial activation and the influx of activated microglia. Since these effects are opposite to injury and neurodegeneration, we propose that they represent adaptive actions of glucocorticoids, preventing cellular defense mechanisms from overshooting. We used adrenalectomy as a model to investigate how adult granule neurons die in vivo and the effects of neurotrophic factors in protecting against apoptosis. Neurotrophin-4/5 and TGF-β1 protected granule neurons against adrenalectomy-induced apoptosis. Since neurogenesis is also greatly increased in the dentate gyrus following adrenalectomy, we compared the time course of birth and death with glial responses. TGF-β1 mRNA increased before the detection of dying cells in the dentate gyrus, which was coincident with increased proliferation in the neurogenic zone. Glucocorticoids also increased Ndrg2 mRNA in glia in the neurogenic zone; Ndrg2 is a member of a novel gene family involved in neural differentiation and synapse formation. Therefore, studying the effects of glucocorticoid manipulation on the dentate gyrus is increasing our understanding of how mature neurons die by apoptosis and the role of glia in induced apoptosis and neurogenesis. Discovering how endocrine and inflammatory responses regulate neuron birth and survival is important for developing successful neuron replacement strategies to treat neurodegenerative diseases.     

Short-term and long-term survival of new neurons in the rat dentate gyrus

New neurons continue to be generated in the dentate gyrus throughout adulthood. Previous studies have shown that a significant proportion of new granule cells labeled with the thymidine analogue bromodeoxyuridine (BrdU) are lost from the adult dentate gyrus within 2 weeks. How long this loss continues and the extent to which it represents cell death, as opposed to dilution of label, is unclear. To address these questions, adult rats were injected with BrdU, and BrdU labeling in the dentate gyrus was compared at several survival time points. Double labeling with BrdU and the cell cycle marker Ki-67 showed that BrdU is detectable for up to 4 days in some cells that continue to divide, indicating that any decrease in the number of BrdU-labeled cells after 4 days is likely to reflect cell death rather than BrdU dilution. Death of new cells in the granule cell layer occurred at a steady rate between 6 and 28 days after labeling, resulting in loss of 50% of BrdU-labeled cells over this 22-day period. New granule cells that survived this first month lived for at least 5 additional months. In contrast, 26% of the granule cells labeled with BrdU at the peak of dentate gyrus development on postnatal day (P) 6 died between 1 and 6 months after labeling. These findings suggest that granule cells born during adulthood that become integrated into circuits and survive to maturity are very stable and may permanently replace granule cells born during development.     

Corticosteroid effects on serotonin responses in granule cells of the rat dentate gyrus

Granule cells in the rat dentate gyrus contain mineralocorticoid and glucocorticoid receptors to which the adrenal hormone corticosterone binds with differential affinity. These cells also express various receptor-subtypes for serotonin (5-HT), including the 5-HT1A receptor which mediates a membrane hyperpolarization accompanied by a decrease in membrane resistance. Earlier studies have shown that removal of corticosterone by adrenalectomy, particularly in the dentate gyrus, results in enhanced expression of the 5-HT1A receptor mRNA and increased 5-HT1A receptor binding capacity. This was normalized by activation of mineralocorticoid receptors or concurrent activation of both receptor types. In the present, intracellular recording study in vitro, we examined if the altered levels of 5-HT1A receptor mRNA and protein are associated with changes in the response to 5-HT. We found that the hyperpolarization and resistance decrease induced in granule cells by a submaximal (10 microM) dose of 5-HT were unaltered 2-4 days after adrenalectomy, indicating a dissociation between corticosteroid actions on 5-HT1A receptor mRNA/protein levels and functional responses to 5-HT. Subsequent occupation of mineralocorticoid receptors in vitro significantly suppressed the 5-HT induced change in resistance, 1-4 h after steroid application. Compared to this, concurrent activation of glucocorticoid receptors led to large responses to 5-HT. This modulation by steroids was not observed with a higher dose of 5-HT (30 microM). The data suggest that with moderate amounts of 5-HT, corticosteroids affect the information flow through the dentate gyrus such that excitatory transmission is promoted with predominant mineralocorticoid receptor activation and attenuated with additional glucocorticoid receptor occupation.     

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.     

Serotonin regulates type II corticosteroid receptor binding in hippocampal cell cultures

Previous work from our laboratory has shown that early postnatal handling of rat pups permanently increases hippocampal type II, but not type I, corticosteroid receptor binding. Handling also increases hippocampal 5-HT turnover, and the effect of handling on type II corticosteroid receptor binding is blocked by concurrent administration of the 5-HT2 receptor antagonist ketanserin. In view of these findings, the present studies examined the effects of 5-HT on type I ([3H]corticosterone) and type II ([3H]RU 28362) corticosteroid receptor binding in dispersed hippocampal cell cultures derived from animals killed at E19-20 in order to verify that 5-HT can act directly on hippocampal cells to alter corticosteroid receptor binding. Both type I and type II receptors were measurable in cultured hippocampal cells and the apparent affinity (Kd) for [3H]corticosterone (0.4 +/- 0.1 nM) and [3H]RU 28362 (0.8 +/- 0.1 nM) was similar to that from studies with intact animals. 5-HT increased type II, but not type I, corticosteroid receptor binding capacity in a dose-related manner, with the maximal effect (+188%) observed at 10 nM 5-HT and no change in the affinity of the receptor for [3H]RU 28362. The effect of 10 nM 5-HT on [3H]RU 28362 binding required a minimum of 4 d exposure and persisted for at least 7 d following the removal of 5-HT. The effect of 10 nM 5-HT on [3H]RU 28362 binding was completely blocked by the 5-HT2 receptor antagonists ketanserin and mianserin. There were no effects of the 5-HT1a antagonist, BMY 7378, or the 5-HT3 antagonist, MDL 72222.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extranuclear estrogen receptor beta immunoreactivity is on doublecortin-containing cells in the adult and neonatal rat dentate gyrus

In adult female rats, estrogen receptor (ER) activation, particularly of ERbeta, promotes hippocampal neurogenesis. We previously reported that extranuclear ERbeta immunoreactivity (ir) in adult rats is on cellular profiles in or near the granule cell layer, which is the location of newly generated cells. During development, cells in or near the granule cell layer transiently express high levels of estrogen binding and nuclear ERs. Thus, we sought to determine if extranuclear ERbeta is in newly generated cells in adult and neonatal rat dentate gyrus. Sections from the dentate gyrus of adult proestrus or postnatal day 7 and 14 female rats were dual-labeled for ERbeta and the new-cell marker doublecortin (DCX) and examined by electron microscopy. DCX-containing neurons were found in the subgranular hilus in adult rats and were more widespread throughout the granule cell layer and hilus of neonatal rats. In both adults and neonatal rats, ERbeta immunoreactivity was found in a subset of DCX-labeled neurons. Electron microscopic examination of the adult dentate gyrus revealed that most perikarya with DCX-ir had the morphological characteristics of granule cells, although a few resembled interneurons. Dendrites with DCX-ir also were observed. In both adults and neonates, DCX-labeled neuronal perikarya and dendrites contained ERbeta-ir; ERbeta-ir usually was aggregated near the plasma membrane, mitochondria or endoplasmic reticula. ERbeta-ir was in glial profiles that apposed DCX-labeled perikarya and dendrites. These findings are consistent with data showing that estrogens can exert non-genomic effects directly and indirectly on newly generated cells in neonatal and adult rat dentate gyrus.     

Increased cell proliferation and granule cell number in the dentate gyrus of protein repair-deficient mice

Recent studies have demonstrated that mice lacking protein L-isoaspartate (D-aspartate) O-methyltransferase (Pcmt1-/- mice) have alterations in the insulin-like growth factor-I (IGF-I) and insulin receptor pathways within the hippocampal formation as well as other brain regions. However, the cellular localization of these changes and whether the alterations might be associated with an increase in cell number within proliferative regions, such as the dentate gyrus, were unknown. In this study, stereological methods were used to demonstrate that these mice have an increased number of granule cells in the granule cell layer and hilus of the dentate gyrus. The higher number of granule cells was accompanied by a greater number of cells undergoing mitosis in the dentate gyrus, suggesting that an increase in neuronal cell proliferation occurs in this neurogenic zone of adult Pcmt1-/- mice. In support of this, increased doublecortin labeling of immature neurons was detected in the subgranular zone of the dentate gyrus. In addition, double immunofluorescence studies demonstrated that phosphorylated IGF-I/insulin receptors in the subgranular zone were localized on immature neurons, suggesting that the increased activation of one or both of these receptors in Pcmt1-/- mice could contribute to the growth and survival of these cells. We propose that deficits in the repair of isoaspartyl protein damage leads to alterations in metabolic and growth-receptor pathways, and that this model may be particularly relevant for studies of neurogenesis that is stimulated by cellular damage.