NTERA-2 Research Articles

Role of zinc in p53‐mediated proliferation and apoptosis of human neuronal precursor cells

Recent reports have established stem cell proliferation and neurogenesis in the adult human brain, primarily localized in the subventricular zone and the subgranular zone of the dentate gyrus. While the trace element zinc is clearly important for normal central nervous system function, no work has explored the role that this essential nutrient plays in adult stem cell proliferation and differentiation. To test this, we employed a model of chelator-induced zinc deficiency in the human stem-cell like neuronal precursor line, Ntera-2 (NT-2). Addition of the zinc chelator TPEN to growth media at concentrations above 7 μM resulted in a significant reduction in cell number compared to untreated control cells. BrdU uptake and labeling, used a marker of cell proliferation, was reduced from approximately 50% in untreated cells over the course of 18 h, to 3% in cells treated with 8 μM TPEN (p<0.001). Additionally, there was both morphological and molecular evidence of apoptosis in TPEN-treated NT-2 cells. Nuclear staining with DAPI showed that zinc deficient cells exhibited classical signs of apoptosis including nuclear shrinkage, chromatin aggregation, and nuclear blebbing. Immunocytochemistry revealed TPEN-induced increases in abundance and nuclear translocation of the tumor suppressor protein p53. Because p53 has been shown to regulate the cell cycle and apoptotic pathways, it is likely that this transcription factor plays a role in the mechanisms regulating zinc deficient NT-2 cells. On-going work is using oligonucleotide array to identify p53-target genes responsible for the regulation of proliferation, survival, and death of neuronal precursors when zinc availability is limited. Supported by FSU Council on Research and Creativity.

Influence of retinoic acid and lithium on proliferation and dopaminergic potential of human NT2 cells

Our laboratory is working with the human NTera2/D1 (NT2) cell line, which has properties similar to those of progenitor cells in the central nervous system (CNS). These neural-like precursor cells can differentiate into all three major lineages, neurons, astrocytes, and oligodendrocytes. The pure neuronal population, hNT neurons, possess characteristics of dopamine (DA) cells. First, we analyzed whether the retinoic acid (RA)-treated hNT neurons and the NT2 precursor cells expressed two transcription factors required for development of the midbrain DA neurons. We report that NT2 cells endogenously expressed Engrailed-1 and Ptx3, whereas RA-treated hNT neurons did not express Engrailed-1 or Ptx3. Next we examined the influence of lithium treatment on Engrailed-1 and Ptx3 as well as another critical transcription factor, Nurr1. Previous research has shown that lithium can mimic the Wnt pathway, which is important for the induction of these transcription factors. Finally, we investigated the effect of lithium treatment on the viability and proliferation of NT2 cells, because lithium has been shown to stimulate neurogenesis in adult neural precursors. Lithium treatment increased the viability and proliferation of NT2 cells. The expression of transcription factors essential for the induction and maintenance of the DA phenotype was not increased in NT2 after lithium treatment. We conclude that the NT2 cell line is an excellent in vitro model system for studying the influence of pharmalogical agents on proliferation, differentiation, and apoptosis of a human neural progenitor cell line.

Delayed Transplantation of Human Neurons following Brain Injury in Rats: A Long-Term Graft Survival and Behavior Study

The NTera2 (NT2) cell line is a homogeneous population of cells, which, when treated in vitro with retinoic acid, terminally differentiate into postmitotic neuronal NT2N cells. Although NT2N neurons transplanted in the acute (24 h postinjury) period survive for up to 1 month following experimental traumatic brain injury (TBI), nothing is known of their ability to survive for longer periods or of their effects when engrafted during the chronic postinjury period. Adult male Sprague-Dawley rats (n = 348; 360-400 g) were initially anesthetized and subjected to severe lateral fluid-percussion (FP) brain injury or sham injury. At 1 month postinjury, only brain-injured animals showing severe neurobehavioral deficits received cryopreserved NT2N neurons stereotaxically transplanted into three sites in the peri-injured cortex (n = 18). Separate groups of similarly brain-injured rats received human fibroblast cells (n = 13) or cell suspension vehicle (n = 14). Sham-injured animals (no brain injury) served as controls and received NT2N transplants (n = 24). All animals received daily immunosuppression for three months. Behavioral testing was performed at 1, 4, 8, and 12 weeks post-transplantation, after which animals were sacrificed for histological analysis. Nissl staining and anti-human neuronal specific enolase (NSE) immunostaining revealed that NT2N neurons transplanted in the chronic post-injury period survived up to 12 weeks post-transplantation, extended processes into the host cortex and immunolabeled positively for synaptophysin. There were no statistical differences in cognitive or motor function among the transplanted brain-injured groups. Long-term graft survival suggests that NT2N neurons may be a viable source of neural cells for transplantation after TBI and also that these grafts can survive for a prolonged time and extend processes into the host cortex when transplanted in the chronic post-injury period following TBI.

Cytotoxic Effects of Low- and High-LET Radiation on Human Neuronal Progenitor Cells: Induction of Apoptosis andTP53Gene Expression

The induction of apoptosis, TP53 expression, caspase activation and cell toxicity were investigated after exposure of cells of the human neuronal progenitor cell line Ntera2 (NT2) to low-LET radiation (gamma and X rays). The data indicates that irradiation of NT2 cells quickly induced TP53 expression, which was followed in time by an increase in caspase activity, and ultimately resulted in the induction of apoptosis. Induction of apoptosis was dependent on dose, and the highest levels were measured 48 h after exposure. For comparison, the level of apoptosis induced by high-LET particle radiation (1 GeV/ nucleon iron ions) was also determined and was found to be dependent on dose. The relative biological effectiveness (RBE) was estimated from the slopes of the dose-response curves for the induction of apoptosis. The RBE(max) for apoptosis 48 h after exposure was at least 3.4. In short, exposure to high-LET radiation results in a more efficient and greater induction of apoptosis in human neuronal progenitor cells than low-LET radiation.

Rapid differentiation of NT2 cells in Sertoli–NT2 cell tissue constructs grown in the rotating wall bioreactor

Cell replacement therapy is of great interest as a long-term treatment of neurodegenerative diseases such as Parkinson's disease (PD). We have previously shown that Sertoli cells (SC) provide neurotrophic support to transplants of dopaminergic fetal neurons and NT2N neurons, derived from the human clonal precursors cell line NTera2/D1 (NT2), which differentiate into dopaminergic NT2N neurons when exposed to retinoic acid. We have created SC-NT2 cell tissue constructs cultured in the high aspect ratio vessel (HARV) rotating wall bioreactor. Sertoli cells, NT2, and SC plus NT2 cells combined in starting ratios of 1:1, 1:2, 1:4 and 1:8 were cultured in the HARV in DMEM with 10% fetal bovine serum and 1% growth factor reduced Matrigel for 3 days, without retinoic acid. Conventional, non-HARV, cultures grown in the same culture medium were used as controls. The presence of tyrosine hydroxylase (TH) was assessed in all culture conditions. Sertoli-neuron-aggregated-cell (SNAC) tissue constructs grown at starting ratios of 1:1 to 1:4 contained a significant amount of TH after 3 days of culture in the HARV. No TH was detected in SC HARV cultures, or SC, NT2 or SC-NT2 conventional co-cultures. Quantitative stereology of immunolabled 1:4 SNAC revealed that approximately 9% of NT2 cells differentiate into TH-positive (TH+) NT2N neurons after 3 days of culture in the HARV, without retinoic acid. SNAC tissue constructs also released dopamine (DA) when stimulated with KCl, suggesting that TH-positive NT2N neurons in the SNAC adopted a functional dopaminergic phenotype. SNAC tissue constructs may be an important source of dopaminergic neurons for neuronal transplantation.

Induction of Cβ splice variants and formation of novel forms of protein kinase A type II holoenzymes during retinoic acid-induced differentiation of human NT2 cells

Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are critical regulators of neuronal differentiation. The expression, levels and activities of PKA subunits were studied prior to and during differentiation of the human neuronal precursor cell line NTera 2 (NT2). Undifferentiated NT2 cells expressed mainly cytoplasmic PKA type I, consisting of the regulatory subunit RIα and the catalytic subunit Cα. Low levels of PKA type II consisting of RIIα or RIIβ associated with Cα were also detected, mainly in the cytoplasm and in the Golgi-centrosomal area. During retinoic acid-induced differentiation, the RIα and RIIα expressions remained in the cytoplasm, while we observed a strong upregulation of RIIβ, located to the whole cytoplasm including neurite extensions. This upregulation coincided with increased PKA-specific activity accompanied by a strong induction of a number of neuronal-specific Cβ splice variants that together with RIIβ form novel PKAII holoenzymes. Formation of novel PKAII holoenzymes may imply specific PKA features which may have consequences for the process of neuronal differentiation and nerve cell function.

The transcription factor Nurr1 in human NT2 cells and hNT neurons

Human, neuronally committed hNT or NT2-N cells, originally derived from the Ntera2/D1 (NT2) clone after exposure to retinoic acid (RA), represent a potentially important source of cells to treat neurodegenerative diseases. Our previous in vitro experiments showed that hNT cells possess immunocytochemically detectable markers typical of dopaminergic (DA) ventral mesencephalic (VM) neurons, including tyrosine hydroxylase (TH), dopamine transporter (DAT), dopamine receptor (D2), and aldehyde dehydrogenase (AHD-2). In the current study, we sought to examine whether Nurr1, an orphan receptor of the nuclear receptor superfamily shown to be essential for the development, differentiation and survival of midbrain DA neurons, would be expressed in 3, 4, or 5 week RA-induced hNT neurons and their NT2 precursors. Our immunocytochemical analyses indicate that NT2 cells as well as hNT neurons independent of the length of RA-driven differentiation were Nurr1-immunoreactive. RT-PCR analysis confirmed the expression of Nurr1-specific mRNA in both NT2 precursors and the hNT neurons. Furthermore, immunocytochemical co-expression of Nurr1 and TH was detected in hNT neurons. The findings of this study suggest that Nurr1 may be important during the development of hNT neurons and involved in their differentiation into the dopaminergic phenotype.

Genetically modified NT2N human neuronal cells mediate long-term gene expression as CNS grafts in vivo and improve functional cognitive outcome following experimental traumatic brain injury.

Human Ntera-2 (NT2) cells can be differentiated in vitro into well-characterized populations of NT2N neurons that engraft and mature when transplanted into the adult CNS of rodents and humans. They have shown promise as treatments for neurologic disease, trauma, and ischemic stroke. Although these features suggest that NT2N neurons would be an excellent platform for ex vivo gene therapy in the CNS, stable gene expression has been surprisingly difficult to achieve in these cells. In this report we demonstrate stable, efficient, and nontoxic gene transfer into undifferentiated NT2 cells using a pseudotyped lentiviral vector encoding the human elongation factor 1-alpha promoter and the reporter gene eGFP. Expression of eGFP was maintained when the NT2 cells were differentiated into NT2N neurons after treatment with retinoic acid. When transplanted into the striatum of adult nude mice, transduced NT2N neurons survived, engrafted, and continued to express the reporter gene for long-term time points in vivo. Furthermore, transplantation of NT2N neurons genetically modified to express nerve growth factor significantly attenuated cognitive dysfunction following traumatic brain injury in mice. These results demonstrate that defined populations of genetically modified human NT2N neurons are a practical and effective platform for stable ex vivo gene delivery into the CNS.

Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), Fort Lauderdale, Florida, May 5-10, 2002.

Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), Fort Lauderdale, Florida, May 5-10, 2002.

Transient modulation of cytoplasmic and nuclear retinoid receptors expression in differentiating human teratocarcinoma NT2 cells.

Human embryonal carcinoma Ntera2/D1 (NT2) cells treated with retinoic acid (RA) differentiate into several cell types including post-mitotic neurons. In this study we asked if RA-induced differentiation alters the expression of RA and retinol (ROL) binding proteins. The regulation of the intracellular carrier proteins for ROL and RA, cellular retinol binding protein I (CRBP-I), and cellular retinoic acid binding protein I and II (CRABP-I, CRABP-II) were studied along with the nuclear RA receptors RARalpha, RARbeta and RARgamma2. PCR analysis of total mRNA from RA-treated cells showed a biphasic early induction of CRBP-I, CRABP-II, and RARgamma2 genes. The immediate early gene Krox-24, a zinc finger transcription factor which is up-regulated during neuronal differentiation, was also induced, but after 1 week of treatment. The induction of CRBP-I protein synthesis in differentiating NT2 cells was confirmed by western blotting and immunofluorescence experiments. Conversely, the synthetic retinoid N-(4-hydroxyphenyl)retinamide, which induces cell death, but not differentiation in different tumour cell types, did not produce the same modulation on gene expression in NT2 cells. These data suggest that the RA-specific induction of CRBP-I and CRABP-II could be an early event in the process leading to neuronal differentiation of NT2 cells.

Clonal Human (hNT) Neuron Grafts for Stroke Therapy: Neuropathology in a Patient 27 Months after Implantation

Although grafted cells may be promising therapy for stroke, survival of implanted neural cells in the brains of stroke patients has never been documented. Human NT2N (hNT) neurons derived from the NTera2 (NT2) teratocarcinoma cell line were shown to remain postmitotic, retain a neuronal phenotype, survive >1 year in host rodent brains and ameliorate motor and cognitive impairments in animal models of ischemic stroke. Here we report the first postmortem brain findings of a phase I clinical stroke trial patient implanted with human hNT neurons adjacent to a lacunar infarct 27 months after surgery. Neurofilament immunoreactive neurons were identified in the graft site, fluorescent in situ hybridization revealed polyploidy in groups of cells at this site just like polyploid hNT neurons in vitro, and there was no evidence of a neoplasm. These findings indicate that implanted hNT neurons survive for >2 years in the human brain without deleterious effects.

Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells.

Inheritance of one of three primary mutations at positions 11778, 3460 or 14484 of the mitochondrial genome in subunits of Complex I causes Leber's Hereditary Optic Neuropathy (LHON), a specific degeneration of the optic nerve, resulting in bilateral blindness. It has been unclear why inheritance of a systemic mitochondrial mutation would result in a specific neurodegeneration. To address the neuron-specific degenerative phenotype of the LHON genotype, we have created cybrids using a neuronal precursor cell line, Ntera 2/D1 (NT2), containing mitochondria from patient lymphoblasts bearing the most common LHON mutation (11778) and the most severe LHON mutation (3460). The undifferentiated LHON-NT2 mutant cells were not significantly different from the parental cell control in terms of mtDNA/nDNA ratio, mitochondrial membrane potential, reactive oxygen species (ROS) production or the ability to reduce Alamar Blue. Differentiation of NT2s resulted in a neuronal morphology and neuron-specific pattern of gene expression, and a 3-fold reduction in mtDNA/nDNA ratio in both mutant and control cells; however, the differentiation protocol yielded significantly less LHON cells than controls, by 30%, indicating either a decreased proliferative potential or increased cell death of the LHON-NT2 cells. Differentiation of the cells to the neuronal form also resulted in significant increases in ROS production in the LHON-NT2 neurons versus controls, which is abolished by rotenone, a specific inhibitor of Complex I. We infer that the LHON genotype requires a differentiated neuronal environment in order to induce increased mitochondrial ROS, which may be the cause of the reduced NT2 yield; and suggest that the LHON degenerative phenotype may be the result of an increase in mitochondrial superoxide which is caused by the LHON mutations, possibly mediated through neuron-specific alterations in Complex I structure.

Nuclear cathepsin B-like protease cleaves transcription factor YY1 in differentiated cells

Differentiation of pluripotent cells into differentiated cell types involves changes in many aspects of cellular biochemistry. Many of these changes result in alterations of gene expression, which may occur by changing the activity of transcription factors. The cell line NTERA-2 (NT2) can be differentiated into various cell types by incubation with retinoic acid. The differentiated cell type is also permissive for infection with the human herpesvirus cytomegalovirus (CMV). The transcription factor YY1 has been shown to regulate the immediate-early promoter of CMV in a differentiation specific manner by binding to one site at −958 to −950 and to at least two sites in the enhancer. It is demonstrated here that there is a second YY1 site in the modulator between −995 and −987. Levels of YY1 DNA binding activity and protein decrease in NT2 cells as they are differentiated with retinoic acid. This decrease in protein is due to the degradation of YY1 by a cathepsin B-like activity found in nuclear extracts. The cleavage products of YY1 include the intact C-terminal half of the protein, which contains the zinc fingers and the DNA binding activity. This suggests a mechanism that allows expression of the CMV immediate-early promoter in differentiated cells.

Chromatin Structure Contributes to Latency

Understanding viral latency and reactivation is an important aspect of viral biology and may aid development of strategies to control viral infection. Meier developed a clonal NTera2/D1 (NT2) cell line, which is derived from an embryonal teratocarcinoma and which can be induced to differentiate into neurons in culture. These cells were used to study latency and reactivation of human cytomegalovirus, a virus that replicates in neurons. Based on analysis of the viral DNA structure in infected, but latent, cells, Meier showed that at least a portion of the viral genome had reached the nucleus. Viral replication could be stimulated by pretreatment of the cells with retinoic acid to stimulate differentiation, but retinoic acid had no effect if applied after infection. The histone deacetylase inhibitor trichostatin A stimulated expression of viral major immediate early genes, viral replication, and the production of infectious virus undifferentiated NT2 cells. Thus, differences in chromatin structure in differentiated and undifferentiated cells represents one mechanism by which the immediate early gene transcription can be inhibited, thus inhibiting viral replication and promoting latency. J. L. Meier, Reactivation of the human cytomegalovirus major immediate-early regulatory region and viral replication in embryonal Ntera2 cells: Role of trichostatin A, retinoic acid, and deletion of the 21-base-pair repeats and modulator. J. Virol. 75 , 1581-1593 (2001). [Abstract] [Full Text]

Chromatin Structure Contributes to Latency

Understanding viral latency and reactivation is an important aspect of viral biology and may aid development of strategies to control viral infection. Meier developed a clonal NTera2/D1 (NT2) cell line, which is derived from an embryonal teratocarcinoma and which can be induced to differentiate into neurons in culture. These cells were used to study latency and reactivation of human cytomegalovirus, a virus that replicates in neurons. Based on analysis of the viral DNA structure in infected, but latent, cells, Meier showed that at least a portion of the viral genome had reached the nucleus. Viral replication could be stimulated by pretreatment of the cells with retinoic acid to stimulate differentiation, but retinoic acid had no effect if applied after infection. The histone deacetylase inhibitor trichostatin A stimulated expression of viral major immediate early genes, viral replication, and the production of infectious virus undifferentiated NT2 cells. Thus, differences in chromatin structure in differentiated and undifferentiated cells represents one mechanism by which the immediate early gene transcription can be inhibited, thus inhibiting viral replication and promoting latency.J. L. Meier, Reactivation of the human cytomegalovirus major immediate-early regulatory region and viral replication in embryonal Ntera2 cells: Role of trichostatin A, retinoic acid, and deletion of the 21-base-pair repeats and modulator. J. Virol. 75, 1581-1593 (2001). [Abstract] [Full Text]

The neuronal repressor REST/NRSF is an essential regulator in medulloblastoma cells.

Medulloblastoma is the most malignant pediatric brain tumor. It is believed to originate from the undifferentiated external granule layer cells in the cerebellum, but the mechanism of tumorigenesis remains unknown. Here we studied three types of human medulloblastoma cells that express markers corresponding to different levels of neuronal differentiation. They expressed the neuronal repressor element 1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF; refs. 7-10) at very high levels compared with either neuronal progenitor NTera2 (NT2) cells or fully differentiated human neuron teratocarcinoma (hNT cells). To counter the effect of REST/NRSF, we used a recombinant transcription factor, REST-VP16, constructed by replacing repressor domains of REST/NRSF with the activation domain of viral protein (VP16). Transient expression of REST-VP16 in medulloblastoma cells was able to compete with the endogenous REST/NRSF for DNA binding and stimulate neuronal promoters. High-efficiency expression of REST-VP16 mediated by adenovirus vectors (Ad.REST-VP16) in medulloblastoma cells was able to counter REST/NRSF-mediated repression of neuronal promoters, stimulate expression of endogenous neuronal genes and trigger apoptosis through the activation of caspase cascades. Furthermore, intratumoral injection of Ad.REST-VP16 in established medulloblastoma tumors in nude mice inhibited their growth. Therefore, REST/NRSF may serve as a new target for therapeutic interventions for medulloblastoma through agents such as REST-VP16.

Differential expression of Bcl-2-related proteins in differentiating NT2 cells.

Although the role of Bcl-2-related proteins as regulators of the apoptotic process has been well documented, recent studies suggest that they might also be implicated in neuronal differentiation. We have studied by immunocytochemistry, Western blotting and RT-PCR the expression pattern of Bcl-xL, Bcl-2 and BAX in the in vitro model of neuronal differentiation constituted by retinoic acid (RA)-treated NTera-2/D1 (NT2/D1) cells. Whereas BAX level did not change significantly during the RA treatment, Bcl-xL level increased markedly during the first week, before returning to basal level during the second week. Bcl-2 expression, undetectable in undifferentiated cells, increased progressively from the first week. From our results, we suggest that, at least in our model, Bcl-2-related proteins might be involved in neuronal differentiation.

Functional characterization of corticotropin-releasing factor type 1 receptor endogenously expressed in human embryonic kidney 293 cells

The endogenous expression in human embryonic kidney 293 (HEK293) cells of corticotropin-releasing factor (CRF) receptors was detected. High-affinity binding sites for human CRF ( K i=3.6 nM), ovine CRF ( K i=4.6 nM), rat urocortin ( K i=2.2 nM), sauvagine ( K i=2.4 nM) and astressin ( K i=4.3 nM) with the pharmacological characteristics for CRF type 1 (CRF 1) receptors and B max values of ∼30 fmol/mg protein were determined. The four CRF receptor agonists nonselectively stimulated cAMP production in HEK293 cells at low agonist concentrations, whereas the antagonist astressin shifted the dose–response curve for ovine CRF significantly rightward. Transfection of the pcDNA3 vector into HEK293 cells strongly reduced the expression of the endogenous CRF receptor. Northern blot analysis revealed the expression of a CRF 1 transcript in human neuronal tissues, HEK293, human NTera-2 (NT2) carcinoma, Y-79 retinoblastoma and African green monkey kidney (COS-7) cells. Neither by Northern blot analysis nor by reverse transcriptase PCR (RT-PCR), the expression of CRF 2 could be detected. In cAMP stimulation experiments, functional CRF receptors were detected in these cell lines. These data show that HEK293 and other cell lines endogenously express CRF 1 receptors.

Differential gene expression between human neurons and neuronal progenitor cells in culture: an analysis of arrayed cDNA clones in NTera2 human embryonal carcinoma cell line as a model system

To elucidate the highly complex expression pattern of the genes involved in human neuronal differentiation, differential gene expression between human neurons and neuronal progenitor cells was investigated by analysis of a cDNA expression array in a pluripotent human embryonal carcinoma cell line NTera2 (NT2), a model system of human neuronal differentiation. Among 588 arrayed cDNA clones, 87 genes showed a differential expression pattern between undifferentiated neuronal progenitor cells (NT2-U) and NT2-derived differentiated neurons induced by treatment with retinoic acid (RA) (NT2-N), while 26 genes could not be analyzed due to high background signals. The levels of expression of 76 genes, including those encoding a group of transcription factors, intracellular signal-transducing proteins, cell death-regulatory proteins, and growth factors/cytokines/neurotransmitters and their receptors, were elevated after neuronal differentiation, while the levels of 11 genes, including those coding for cellular proliferation-related proteins, were decreased. Among the differentially expressed genes following induction of neuronal differentiation, significant up-regulation of the growth-associated protein (GAP-43), low-affinity nerve growth factor receptor p75 (LNGFR), and defender against apoptotic cell death (DAD1) mRNAs and substantial down-regulation of the proliferation-associated gene (PAG), fibroblast growth factor receptor-1 (FGFR-1), and cellular RA-binding protein-II (CRABP-II) mRNAs were verified by Northern blot analysis. These results indicate that the analysis of cDNA expression arrays provides a useful approach for screening and identification of a set of distinct genes that undergo highly complex regulation during human neuronal differentiation.

GABAA Receptors Expressed in Undifferentiated Human Teratocarcinoma NT2 Cells Differ from Those Expressed by Differentiated NT2-N Cells

During CNS development, changes occur in expression of GABA(A) receptor subunit subtypes and GABA(A) receptor pharmacological and biophysical properties. We used reverse transcription PCR and whole-cell-recording techniques to determine whether GABA(A) receptor expression and function also changed during retinoic acid-induced differentiation of human Ntera 2 (NT2) teratocarcinoma cells into neuron-like cells (NT2-N cells). In undifferentiated NT2 cells only alpha5, beta3, gamma3, and pi subtype mRNAs were detected. NT2 GABA(A) receptor currents had a maximal amplitude of 52 pA and an EC(50) of 4.0 microM, were relatively insensitive to enhancement by zolpidem and diazepam, and were enhanced by loreclezole and inhibited by lanthanum, zinc, and furosemide. In contrast, in NT2-N cells after 13 weeks of retinoic acid treatment, all GABA(A) receptor subtype mRNAs were detected. Maximal peak whole-cell currents were approximately 50-fold larger than NT2 cell currents, and the GABA EC(50) was higher (39.7 microM). In 13 week NT2-N cells, diazepam, zolpidem, loreclezole, and lanthanum had only small effects on GABA(A) receptor currents, and the zinc IC(50) for current inhibition was significantly higher than that for NT2 cells. In a previous study, we showed that NT2-N cells after 5 weeks of retinoic acid treatment had moderate peak currents, GABA EC(50,) and zinc IC(50) but that currents were robustly enhanced by diazepam, zolpidem, and loreclezole. During differentiation of NT2 cells to NT2-N cells, GABA(A) receptors underwent changes in subunit expression and pharmacology that were similar to many of the developmental changes in GABA(A) receptors that occur in CNS neurons.