NGF stimulation increases JNK2 phosphorylation and reduces caspase-3 activity in the olfactory bulb of estrogen-replaced animals.

Estrogen receptors are extensively colocalized with neurotrophins and their receptors in the rodent forebrain. We have shown previously that estrogen increases mRNA and protein expression of the nerve growth factor (NGF)-specific tyrosine kinase receptor, trkA, while decreasing expression of the universal neurotrophin receptor p75. In view of the pro-survival roles described for trks and the context-dependent stimulation of survival and cell death pathways activated by p75, differential regulation of these receptors by estrogen is likely to alter neurotrophin-dependent cell signaling. This hypothesis was tested in vivo, using the rodent olfactory bulb as a model. We found that NGF activated the extracellular signal-regulated protein kinase (ERK) equally in estrogen replaced and hormone-deprived animals. However in the case of c-jun-kinase (JNK), a related MAP kinase, pretreatment with estrogen altered NGF activation of a specific isoform of this protein. Specifically, NGF stimulation did not alter JNK1 or JNK2 activation in the estrogen-deprived condition, but significantly increased JNK2 activation in estrogen-replaced animals. Increased JNK2 phosphorylation in the NGF-injected, estrogen- replaced animals was paralleled by decreased activity of caspase-3, an enzyme required for apoptosis. In view of the disparate roles assigned to JNK, this latter finding suggests that estrogen pretreatment may preferentially direct neurotrophin-dependent JNK activation toward regeneration and plasticity rather than cell death.

Neurotrophin expression in the reproductively senescent forebrain is refractory to estrogen stimulation.

The present studies compared the regulation of the neurotrophin ligands and receptors by estrogen in young adult and reproductively senescent rats. Both groups of animals were ovariectomized and replaced with 17beta-estradiol or placebo pellets for 4 weeks. Protein expression of specific neurotrophins and their receptors were measured in the olfactory bulb and its basal forebrain afferent, the horizontal limb of the diagonal band of Broca (hlDBB). Young-adult rats responded to estrogen with an increase in the expression of brain-derived neurotrophic factor (BDNF) in the olfactory bulb and hlDBB, as well as bulbar trkA and trkB receptors. Older rats did not respond to estrogen in this manner. Additionally, estrogen treatment decreased the expression of the universal neurotrophin receptor p75 in young adult animals, but increased expression of this receptor in reproductively senescent rats. The latter group, however, had significantly greater estrogen receptor alpha (ERalpha) expression in the olfactory bulb as compared to their younger counterparts, but very low expression of the steroid receptor coactivator, SRC-1. Changes in the proportion or ratio of steroid receptor/coactivator systems in the aging forebrain may contribute to the refractory response to estrogen in the reproductively senescent animals.

Region- and peptide-specific regulation of the neurotrophins by estrogen.

We have previously shown that estrogen increases the expression of brain-derived neurotrophic factor (BDNF) mRNA in the olfactory bulb and cingulate cortex. Here we report that estrogen regulation of BDNF protein and the structurally related peptides nerve growth factor (NGF) and neurotrophin (NT)-4 is region- and peptide-specific. The olfactory bulb and cingulate cortex are both estrogen-sensitive targets and each receives a separate projection from neurons in the horizontal limb of the diagonal band of Broca (hlDBB). Furthermore, neurotrophins are retrogradely transported from the bulbar and cortical targets to the hlDBB. Four weeks of estrogen replacement to ovariectomized animals increased BDNF expression in the olfactory bulb, but decreased BDNF in the cingulate cortex. On the other hand, estrogen increased NT-4 expression in the cingulate cortex, but not in the olfactory bulb. NGF expression was not affected by estrogen in either region studied. In the hlDBB, estrogen increased BDNF but decreased NT-4, suggesting that estrogen differentially affects retrograde accumulation of these peptides. While both estrogen receptor alpha and beta have been identified in the olfactory bulb and cingulate cortex, our results indicate that estrogen receptor alpha expression is relatively higher in the olfactory bulb as compared to the cortex. Since the two estrogen receptors have been shown to stimulate different signaling pathways, we hypothesize that estrogen acting through specific receptors may differentially influence the extent and direction of neurotrophin expression.

Projections of androgen-accumulating neurons in a nucleus controlling avian song.

In zebra finches, androgens stimulate song production and promote growth of the neural regions controlling song. Early exposure to estrogen establishes this sensitivity to androgens and increases the number of androgen-accumulating cells in two song regions, the hyperstriatum ventralis pars caudalis (HVc) and the magnocellular nucleus of the anterior neostriatum. To examine if these regional changes in androgen accumulation could directly influence androgen responsiveness elsewhere in the song system, we combined autoradiographic and retrograde tracing techniques to determine if androgen-accumulating HVc neurons project to other vocal control nuclei. We report here that both major efferent projections from HVc (to Area X and the nucleus robustus of the archistriatum) include a substantial proportion of androgen-accumulating neurons. These data are consistent with the hypothesis that the extent of androgen accumulation in HVc may in turn regulate the androgenic sensitivity of other song regions.

Characterization of neurons born and incorporated into a vocal control nucleus during avian song learning.

In male zebra finches, song learning is accompanied by the addition of new neurons to Area X, a nucleus necessary for normal song development. We examined whether Area X neurons born after 15 days post-hatch are recruited into the efferent pathway from Area X to the dorsolateral nucleus of the medial anterior thalamus (DLM). Our data suggest that the majority of these new Area X neurons are interneurons and that DLM-projecting neurons are incorporated into Area X prior to song learning.

Stroke neuroprotection: oestrogen and insulin-like growth factor-1 interactions and the role of microglia.

Oestrogen has been shown to be neuroprotective for stroke and other neural injury models. Oestrogen promotes a neuroprotective phenotype through myriad actions, including stimulating neurogenesis, promoting neuronal differentiation and survival, suppressing neuroinflammation and maintaining the integrity of the blood-brain barrier. At the molecular level, oestrogen directly modulates genes that are beneficial for repair and regeneration via the canonical oestrogen receptor. Increasingly, evidence indicates that oestrogen acts in concert with growth factors to initiate neuroprotection. Oestrogen and insulin-like growth factor (IGF)-1 act cooperatively to influence cell survival, and combined steroid hormone/growth factor interaction has been well documented in the context of neurones and astrocytes. Here, we summarise the evidence that oestrogen-mediated neuroprotection is critically dependent on IGF-1 signalling, and specifically focus on microglia as the source of IGF-1 and the locus of oestrogen-IGF-1 interactions in stroke neuroprotection.

Nerve growth factor (NGF) regulation of estrogen receptors in explant cultures of the developing forebrain.

Estrogen profoundly affects the organization of the nervous system. Its receptor, a nuclear transcription factor, is widely expressed in the developing forebrain. Earlier work established that forebrain estrogen target neurons coexpress nerve growth factor (NGF) receptors and receptor mRNA. The present study examined the regulation of forebrain estrogen receptors by NGF, using organotypic cultures of the developing cerebral cortex and basal forebrain. NGF significantly increased nuclear estrogen binding in cortical but not basal forebrain explants. Both cortical and basal forebrain explant cultures express the NGF receptor, TrkA. However, our earlier observation that developing cortical neurons, unlike basal forebrain neurons, widely coexpress mRNAs for NGF and its cognate receptors, suggests that in the present study cortical neuronal responses to exogenous NGF may have been primed by autocrine mechanisms. Alterations in nuclear estrogen binding but not estrogen receptor mRNA levels suggests that NGF may regulate cortical estrogen receptors posttranscriptionally.

Estrogen differentially regulates estrogen and nerve growth factor receptor mRNAs in adult sensory neurons.

We have previously shown that neurons in the basal forebrain colocalize the neurotrophin receptor p75NGFR and estrogen receptors. The present study was designed to examine (1) if neural neurotrophin targets respond to estrogen as a general phenotypic feature and (2) if NGF receptor mRNAs are regulated by estrogen, using a prototypical target of NGF, the dorsal root ganglion (DRG) (sensory) neuron. We demonstrate, for the first time, the presence of estrogen receptor mRNA and protein (binding sites) in adult female rat DRG. Moreover, estrogen receptor mRNA expression, while present in DRG neurons from both the ovariectomized (OVX; estrogen deficient) and intact female rat, was downregulated, as in the adult CNS, during proestrus (high estrogen levels) and in OVX animals replaced with proestrus levels of estrogen, as compared to OVX controls. In contrast, although the mRNAs for the NGF receptors p75NGFR and trkA were also expressed in DRG neurons from OVX and intact animals, expression of both NGF receptor mRNAs was upregulated in sensory neurons during proestrus, as compared to the OVX condition. Estrogen replacement, on the other hand, resulted in a transient downregulation of p75NGFR mRNA and a time-dependent upregulation of trkA mRNA. Estrogen regulation of NGF receptor mRNA in adult peripheral neural targets of the neurotrophins supports the hypothesis that estrogen may regulate neuronal sensitivity to neurotrophins such as NGF and may be an important mediator of neurotrophin actions in normal neural function and following neural trauma.

Identification of a putative estrogen response element in the gene encoding brain-derived neurotrophic factor.

We have been studying the role and mechanism of estrogen action in the survival and differentiation of neurons in the basal forebrain and its targets in the cerebral cortex, hippocampus, and olfactory bulb. Previous work has shown that estrogen-target neurons in these regions widely coexpress the mRNAs for the neurotrophin ligands and their receptors, suggesting a potential substrate for estrogen-neurotrophin interactions. Subsequent work indicated that estrogen regulates the expression of two neurotrophin receptor mRNAs in prototypic peripheral neural targets of nerve growth factor. We report herein that the gene encoding the neurotophin brain-derived neurotrophic factor (BDNF) contains a sequence similar to the canonical estrogen response element found in estrogen-target genes. Gel shift and DNA footprinting assays indicate that estrogen receptor-ligand complexes bind to this sequence in the BDNF gene. In vivo, BDNF mRNA was rapidly up-regulated in the cerebral cortex and the olfactory bulb of ovariectomized animals exposed to estrogen. These data suggest that estrogen may regulate BDNF transcription, supporting our hypothesis that estrogen may be in a position to influence neurotrophin-mediated cell functioning, by increasing the availability of specific neurotrophins in forebrain neurons.

Selective impairment of song learning following lesions of a forebrain nucleus in the juvenile zebra finch.

Area X, a large sexually dimorphic nucleus in the avian ventral forebrain, is part of a highly discrete system of interconnected nuclei that have been implicated in either song learning or adult song production. Previously, this nucleus has been included in the song system because of its substantial connections with other vocal control nuclei, and because its volume is positively correlated with the capacity for song. In order to directly assess the role of Area X in song behavior, this nucleus was bilaterally lesioned in both juvenile and adult zebra finches, using ibotenic acid. We report here that lesioning Area X disrupts normal song development in juvenile birds, but does not affect the production of stereotyped song by adult birds. Although juvenile-lesioned birds were consistently judged as being in earlier stages of vocal development than age-matched controls, they continued to produce normal song-like vocalizations. Thus, unlike the lateral magnocellular nucleus of the anterior neostriatum, another avian forebrain nucleus implicated in song learning, Area X does not seem to be necessary for sustaining production of juvenile song. Rather, the behavioral results suggest Area X is important for either the acquisition of a song model or the improvement of song through vocal practice.

Local and cortical effects of olfactory bulb lesions on trophic support and cholinergic function and their modulation by estrogen.

This study determined whether olfactory bulb lesions would affect trophic support to its afferent, the horizontal limb of the diagonal band of Broca (hIDBB), and if estrogen would ameliorate the effects of neural injury in this circuit. NMDA injections into the olfactory bulb resulted in neural injury as indicated by cell loss and increased glial fibrillary acidic protein immunoreactivity. Olfactory bulb lesions severely reduced BDNF expression in its afferent, the hIDBB, while NGF was only reduced in lesioned animals deprived of estrogen. In the olfactory bulb itself, lesions increased BDNF expression, but not NGF. Paradoxically, bulb lesions up-regulated both NGF and BDNF in another target of the hIDBB, the cingulate cortex. Moreover, olfactory bulb lesions affected choline uptake and ChAT activity locally, as well as in the cingulate cortex. Estrogen significantly attenuated the lesion-induced loss of choline uptake in the cingulate cortex, but not at the primary lesion site. Collectively, these results indicate that neural injury to one limb of the forebrain cholinergic system may result in collateral damage to other limbs of this system, suggesting a mechanism for the progression of neurodegenerative diseases, such as Alzheimer's disease, that involve the cholinergic system. Furthermore, these data also indicate that estrogen selectively attenuates certain lesion-induced deficits.

Neuronal colocalization of mRNAs for neurotrophins and their receptors in the developing central nervous system suggests a potential for autocrine interactions.

Development and survival of neurons in the central nervous system are dependent on the activity of a variety of endogenous neurotrophic agents. Using combined isotopic and nonisotopic in situ hybridization histochemistry, we have found that subsets of neurons within the developing forebrain coexpress the mRNAs for both neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3) and their receptors (p75NGFR, TrkA, and TrkB). The colocalization of mRNA for neurotrophin receptors and their ligands in presumptive neurotrophin target neurons suggests the potential for autocrine and paracrine mechanisms of action during development. Such mechanisms may ensure the onset of differentiation and survival of specific subsets of neurons prior to and following target innervation.

Interactions of estrogen with the neurotrophins and their receptors during neural development.

We are interested in examining mechanisms underlying estrogen actions during neuronal differentiation in the central nervous system (CNS). Our research has focused on one possible mechanism, the developmental interactions between estrogen and the neurotrophins (nerve growth factor [NGF], brain derived neurotrophic factor [BDNF] and neurotrophin-3 [NT-3]). Using combined isotopic and non-isotopic in situ hybridization, we found that neurons in developmental estrogen targets (e.g., the cerebral cortex), co-localized mRNAs for the neurotrophins (NGF or BDNF) with their cognate receptors (p75NGFR [the pan-neurotrophin receptor] and trkA or trkB [the tyrosine kinase receptors]), suggesting a localization of neurotrophin-autocrine loops to these estrogen-sensitive neurons. In contrast, the basal forebrain, which is estrogen-sensitive in the adult and during development, only expressed neurotrophin receptor mRNAs, suggesting that this region was not an autocrine neurotrophin target. We examined the potential for developmental estrogen-neurotrophin interactions, using a model neurotrophin-sensitive system, i.e., differentiating PC12 cells. NGF significantly increased estrogen receptor density in PC12 cells. Reciprocally, estrogen up-regulated trkA mRNA and transiently down-regulated p75NGFR mRNA, suggesting that estrogen may increase the efficiency of NGF binding in PC12 cells. Similar estrogen-dependent regulation of NGF receptor mRNAs were also observed in the adult dorsal root ganglia, suggesting that estrogen may regulate NGF sensitivity in adult neurotrophin targets as well. Such estrogen-neurotrophin interactions may have an important role during differentiation and in the adult, following injury.

Presumptive Estrogen Target Neurons Express mRNAs for both the Neurotrophins and Neurotrophin Receptors: A Basis for Potential Developmental Interactions of Estrogen with the Neurotrophins.

Estrogen and the neurotrophins regulate development, survival, and plasticity of the nervous system. We have shown previously that neurons of the developing basal forebrain and their cortical and hippocampal targets express estrogen receptor mRNA and protein. Furthermore, subsets of neurons within these regions colocalize mRNAs for neurotrophin receptors (p75(NGFR), (trk) A, and (trk)B) and their cognate ligands (NGF, BDNF, and NT-3). Using combined isotopic/nonisotopic in situ hybridization histochemistry, we now demonstrate that mRNAs for the neurotrophins as well as their receptors colocalize to individual estrogen receptor mRNA-containing neurons in these regions of the developing rodent forebrain. The patterns of colocalization were both region and mRNA specific. These results suggest a potential for interactions between estrogen and the neurotrophins, including possible estrogen-stimulated, neurotrophin-mediated autocrine mechanisms that may regulate neuronal differentiation and survival during development.

Expression of brain-derived neurotrophic factor and its cognate receptor, TrkB, in the rat suprachiasmatic nucleus.

Photic entrainment of mammalian circadian rhythms occurs because the pacemaker in the suprachiasmatic nuclei (SCN) of the hypothalamus is endowed with a rhythmic sensitivity to photic signals conveyed by the retinohypothalamic tract. Since brain-derived neurotrophic factor (BDNF) has been implicated in the functional modulation of other retinal targets, the rat SCN was examined for expression and cellular distribution of this neurotrophin and TrkB, the tyrosine kinase receptor that preferentially binds BDNF. The rat SCN was found to express the mature BDNF peptide and mRNA by Western blotting, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR) analyses. BDNF-immunoreactivity and hybridization signal for its mRNA were coextensively localized within a number of SCN cells throughout the rostrocaudal axis of each nucleus. In addition, some cells intercalated within the optic chiasm were distinguished by expression of BDNF immunoreactivity or mRNA. Immunostaining for the TrkB receptor was also evident in the SCN within terminals or fibers predominantly located along the SCN/optic chiasm interface and within scattered perikarya near the medial border of each nucleus. Combined in situ hybridization and immunocytochemical analysis revealed that BDNF mRNA-expressing cells within the ventrolateral SCN were often closely apposed to TrkB-positive fibers extending from the optic chiasm. These findings raise the possibility that target-derived interactions between BDNF and TrkB receptors could play a role in the circadian modulation of SCN pacemaker sensitivity to photic input transmitted by the retinohypothalamic tract.

Developmental and hormonal regulation of NR2A mRNA in forebrain regions controlling avian vocal learning.

Developmental changes in the composition of NMDA receptors can alter receptor physiology as well as intracellular signal transduction cascades, potentially shifting thresholds for neural and behavioral plasticity. During song learning in zebra finches, NMDAR currents become faster, and transcripts for the modulatory NR2B subunit of this receptor decrease in lMAN, a region in which NMDAR activation is critical for vocal learning. Using in situ hybridization, we found that NR2A transcripts change reciprocally, increasing significantly in both lMAN (59%) and in another song region, Area X (38%), between posthatch day (PHD) 20 and 40, but not changing further at PHD60 or 80. In adjacent areas not associated with song learning, NR2A mRNA did not change between PHD20-80. Although early song deprivation (which extends the sensitive period for song learning) delays changes in NR2B gene expression and NMDAR physiology within the lMAN, it did not alter NR2A mRNA levels measured at PHD40, 45, or 60. Early testosterone (T) treatment, which disrupts vocal development and accelerates the maturation of both NR2B levels and NMDAR physiology in lMAN, also significantly increased NR2A transcripts measured at PHD35 in lMAN. In Area X, a similar effect of T approached significance. Together with our previous studies, these results show that in a pathway critical for vocal plasticity, the ratio of NR2A:NR2B mRNA rises abruptly early during the sensitive period for song learning. Furthermore, androgen regulation of NMDAR gene expression may alter thresholds for experience-dependent synaptic change.

Developmental regulation of NMDA receptor 2B subunit mRNA and ifenprodil binding in the zebra finch anterior forebrain.

In passerine songbirds, song learning often is restricted to an early sensitive period and requires the participation of several discrete regions within the anterior forebrain. Activation of N-methyl-D-aspartate (NMDA) receptors is implicated in song learning and in one forebrain song region, the lateral magnocellular nucleus of the anterior neostriatum (IMAN), NMDA receptors decrease in density, their affinity for the antagonist MK-801 increases, and their currents decay more quickly as young male zebra finches lose the ability to imitate new song elements. These developmental changes in NMDA receptor pharmacology and physiology suggest that the subunit composition of NMDA receptors changes developmentally. Here, we have used in situ hybridization and [3H]ifenprodil receptor autoradiography to study the developmental regulation of the NMDA receptor 2B subunit (NR2B) within the anterior forebrain of male zebra finches. NR2B mRNA expression within the IMAN was twice as great in 30-day-old males (early in the sensitive period for song learning) as in adult males, and this developmental decrease in NR2B mRNA expression was mirrored by a decrease in high-affinity (NR2B-associated) [3H]ifenprodil binding within this song region. In another anterior forebrain song region, Area X, NR2B mRNA also declined significantly after 30 days posthatch, but this decline was not accompanied by a significant decrease in [3H]ifenprodil binding. The results are consistent with the hypothesis that developmental changes in NMDA receptor function mediated by regulation of subunit composition contribute to the sensitive period for vocal learning in birds.

Reciprocal regulation of estrogen and NGF receptors by their ligands in PC12 cells.

Recent work has shown that estrogen receptor mRNA and protein co-localize with neurotrophin receptor systems in the developing basal forebrain. In the present study we examined the potential for reciprocal regulation of estrogen and neurotrophin receptor systems by their ligands in a prototypical neurotrophin target, the PC12 cell. Using in situ hybridization histochemistry, RT-PCR and a modified nuclear exchange assay, we found both estrogen receptor mRNA and estrogen binding in PC12 cells. Moreover, while estrogen binding was relatively low in naive PC12 cells, long-term exposure to NGF enhanced estrogen binding in these cells by sixfold. Furthermore, concurrent exposure to estrogen and NGF differentially regulated the expression of the two NGF receptor mRNAs. The expression of trkA mRNA was up-regulated, while p75NGFR mRNA was down-regulated transiently. The present data indicate that NGF may increase neuronal sensitivity to estrogen, and that estrogen, by differentially regulating p75NGFR and trkA mRNA, may alter the ratio of the two NGF receptors, and, consequently, neurotrophin responsivity. In view of the widespread co-localization of estrogen and neurotrophin receptor systems in the developing CNS, the reciprocal regulation of these receptor systems by NGF and estrogen may have important implications for processes governing neural maturation and the maintainance of neural function.

Alcohol exposure during the first two trimesters equivalent alters granule cell number and neurotrophin expression in the developing rat olfactory bulb.

Although alcohol has been shown to affect brain development adversely, the underlying mechanism of alcohol's actions are poorly understood. The present study addressed the hypothesis that alcohol affects growth factor availability during critical periods of neural growth by measuring the mRNA expression of brain-derived neurotrophic factor (BDNF), a potent developmental growth factor. Multiple offspring of timed-pregnant rat dams given alcohol (6.0 g/kg per day) or control treatments during gestation were sacrificed at either embryonic (E) day 21 or E33 (usually postnatal day 10) when their olfactory bulbs were processed for molecular analyses or neuron counting. BDNF mRNA levels were measured by reverse-transcription-polymerase chain reaction, and DNA methylation of the BDNF gene was quantified by Southern blot analyses following digestion with methylation-sensitive enzymes. Estimates of total granule cell number were obtained by counting those cells using unbiased stereological techniques. There was a significant decrease in BDNF mRNA levels in the alcohol-exposed offspring of both ages compared with controls. In addition, the number of olfactory bulb granule cells significantly decreased in the E33 but not the E21 rat pups exposed to alcohol compared with their appropriate aged controls. Finally, BDNF DNA of alcohol-exposed animals was less susceptible to digestion with the methylation-sensitive enzyme HpaII compared with controls, suggesting that the DNA of the alcohol exposed pups was hypermethylated. Our results indicate that exposure to alcohol during early brain development in the rat, a period equivalent to the first two trimesters in humans, can have a detrimental effect on normal development of the olfactory bulb by reducing the number of BDNF-synthesizing neurons. Although the exact mechanism for the alcohol-induced neuronal loss is unknown, the inappropriate transcription of the BDNF gene is one mechanism that may account for the complexity of effects observed in offspring exposed to heavy alcohol exposure in utero.

Fas/Apo [apoptosis]-1 and associated proteins in the differentiating cerebral cortex: induction of caspase-dependent cell death and activation of NF-kappaB.

The developing cerebral cortex undergoes a period of substantial cell death. The present studies examine the role of the suicide receptor Fas/Apo[apoptosis]-1 in cerebral cortical development. Fas mRNA and protein are transiently expressed in subsets of cells within the developing rat cerebral cortex during the peak period of apoptosis. Fas-immunoreactive cells were localized in close proximity to Fas ligand (FasL)-expressing cells. The Fas-associated signaling protein receptor interacting protein (RIP) was expressed by some Fas-expressing cells, whereas Fas-associated death domain (FADD) was undetectable in the early postnatal cerebral cortex. FLICE-inhibitory protein (FLIP), an inhibitor of Fas activation, was also expressed in the postnatal cerebral cortex. Fas expression was more ubiquitous in embryonic cortical neuroblasts in dissociated culture compared to in situ within the developing brain, suggesting that the environmental milieu partly suppresses Fas expression at this developmental stage. Furthermore, FADD, RIP, and FLIP were also expressed by subsets of dissociated cortical neuroblasts in culture. Fas activation by ligand (FasL) or anti-Fas antibody induced caspase-dependent cell death in primary embryonic cortical neuroblast cultures. The activation of Fas was also accompanied by a rapid downregulation of Fas receptor expression, non-cell cycle-related incorporation of nucleic acids and nuclear translocation of the RelA/p65 subunit of the transcription factor NF-kappaB. Together, these data suggest that adult cortical cell number may be established, in part, by an active process of receptor-mediated cell suicide, initiated in situ by killer (FasL-expressing) cells and that Fas may have functions in addition to suicide in the developing brain.