P11 expression and PET in bipolar disorders.

BACKGROUND: Bipolar disorder (BD) is a common mental disorder, subdivided into BD-I and BD-II. Currently, few biomarkers differentiate BD-I from BD-II. However, it is suggested that peripheral blood mononuclear cell (PBMC) mRNA levels of p11 and positron emission tomography (PET) might be potential biomarkers for BD. METHODS: Healthy controls (HCs), BD-I, and BD-II patients in remission (n = 20 in each group) underwent a resting PET study with the radiotracer [(18)F]-2-deoxy-2-fluoro-d-glucose ((18)F-FDG). PBMC p11 mRNA levels were determined by quantitative real-time PCR. RESULTS: Comparing BD patients to HCs, normalized glucose metabolism (NGM) was higher in the hippocampus, parahippocampus, and amygdala, but lower in the anterior cingulate cortex (aCC), medial prefrontal cortex (mPFC), dorsolateral prefrontal cortex (dlPFC), insula and thalamus. Compared to BD-II, BD-I had hypometabolism of glucose in the aCC, bilateral middle and inferior gyrus, insula and striatum, and hypermetabolism of glucose in the left parahippocampus. PBMC p11 mRNA was over-expressed in both BD-I and BD-II, although there was no significant difference in its expression levels between BD-I and B-II patients. Further, there were significant positive correlations between PBMC p11 mRNA and NGM in the mPFC, aCC, left insula, bilateral orbitofrontal cortex (OFC), and left middle, inferior and superior temporal gyri. Also, PBMC p11 mRNA was positively correlated to the number of depressive episodes in BD patients, especially in BD-I patients. DISCUSSION: This study demonstrates that PBMC p11 mRNA expression is associated with neural activation in the brain of BD patients and warrants a larger translational study to determine its clinical utility.

P11 (S100A10) as a potential biomarker of psychiatric patients at risk of suicide.

Although suicide represents 1.8% of the global burden of disease, there are few objective assays for suicide risk. Being associated with depressive disorders, which have a high risk of suicide, the proteins P11, P2RX7, and S100ß may be biomarkers for a suicidal disposition. We measured levels of p11 and P2RX7 mRNA in peripheral blood mononuclear cells (PBMCs) of 26 psychiatric patients (11 suicide attempters, 15 suicide non-attempters) with post-traumatic stress disorder (PTSD) and major depressive disorder (MDD), and 14 normal controls, using quantitative real-time PCR. We also conducted a meta-analysis of microarray data of p11, P2RX7 and S100ß from post-mortem prefrontal cortex (PFC) of patients who committed suicide (n = 56) and non-suicide controls (n = 61). We found that PBMC p11 mRNA levels were significantly lower in suicide attempters and higher in suicide non-attempters, when compared to normal controls. The PFC p11 mRNA levels in suicide completers were also lower than non-suicide controls (adjusted p = 0.007). Unlike p11, PBMC P2RX7 mRNA levels were significantly lower than normal controls in all patients including suicide attempters, suicide non-attempters, and suicide completers. In addition, levels of S100ß in PFC did not differ between suicide completers and non-suicide controls. These results suggest that PBMC p11 mRNA levels may be a potential adjunctive biomarker for the assessment of suicide risk in mental disorders and warrants a larger translational study to determine its clinical utility.

Integrating microRNA and mRNA expression profiles of neuronal progenitors to identify regulatory networks underlying the onset of cortical neurogenesis.

BACKGROUND: Cortical development is a complex process that includes sequential generation of neuronal progenitors, which proliferate and migrate to form the stratified layers of the developing cortex. To identify the individual microRNAs (miRNAs) and mRNAs that may regulate the genetic network guiding the earliest phase of cortical development, the expression profiles of rat neuronal progenitors obtained at embryonic day 11 (E11), E12 and E13 were analyzed. RESULTS: Neuronal progenitors were purified from telencephalic dissociates by a positive-selection strategy featuring surface labeling with tetanus-toxin and cholera-toxin followed by fluorescence-activated cell sorting. Microarray analyses revealed the fractions of miRNAs and mRNAs that were up-regulated or down-regulated in these neuronal progenitors at the beginning of cortical development. Nearly half of the dynamically expressed miRNAs were negatively correlated with the expression of their predicted target mRNAs. CONCLUSION: These data support a regulatory role for miRNAs during the transition from neuronal progenitors into the earliest differentiating cortical neurons. In addition, by supplying a robust data set in which miRNA and mRNA profiles originate from the same purified cell type, this empirical study may facilitate the development of new algorithms to integrate various "-omics" data sets.

Levels of the potential biomarker p11 in peripheral blood cells distinguish patients with PTSD from those with other major psychiatric disorders.

Posttraumatic stress disorder (PTSD) is a severely debilitating anxiety disorder. Over 80% of patients with PTSD also exhibit other psychiatric condition, such as bipolar disorder (BP) or major depression (MDD). Previously, it has been found that p11 mRNA expression was significantly changed in post mortem cortex of patients with PTSD and depression. We hypothesize that p11 mRNA levels in the peripheral blood cells will be a potential biomarker for PTSD with heterogeneity in terms of type of trauma, time since trauma and duration of illness. We examined the peripheral blood mononuclear cell (PBMC) P11 mRNA of patients with PTSD (n=13), major depressive disorder (MDD, n=16), bipolar disorder (BP, n=24), and schizophrenia (SCZ, n=12) or controls (n=14) using quantitative real-time PCR and the circulating levels of cortisol in blood plasma and saliva of PTSD using radioimmunoassay kit CORT-CT2. The Hamilton Rating Scale for Depression (HAMD) and Anxiety (HARS), the Chinese version of the Davidson Trauma Scale-Frequency (CDTS-F) and the Chinese version of the Davidson Trauma Scale-Severity (CDTS-S), and Impact of Event Scale-Revised (IES-R) were administered. We found that patients with PTSD had lower levels of p11 mRNA than control subjects, while those with MDD, BP and SCZ had significantly higher p11 levels than the controls. P11 mRNA levels were positively correlated with the scores of HAMD (r=0.62, p<0.05), CDTS-F (r=0.71, p<0.05) and CDTS-S (r=0.62, p<0.05), while they did not correlate with scores of HARS and IES-R. Basal levels of plasma and salivary cortisol of PTSD patients were not statistically different from those of controls. Our findings suggest that PBMC p11 mRNA expression levels may serve as a potential biomarker to distinguish PTSD from BP, MDD and SCZ.

Reconstruction of functional cortical-like tissues from neural stem and progenitor cells.

Neural stem and progenitor cells isolated from embryonic day 13 rat cerebral cortex were immobilized in three-dimensional type I collagen gels, and then the cell-collagen constructs were transferred to rotary wall vessel bioreactors and cultured in serum-free medium containing basic fibroblast growth factor (bFGF) combined with brain-derived neurotrophic factor for up to 10 weeks. Remarkably, the collagen-entrapped cells formed a complex two-layered structure that emulated to a certain extent the cerebral cortex of the embryonic brain in architecture and functionality. The surface layer (layer I) composed primarily of proliferating neural progenitor cells (nestin(+), vimentin(+), and PCNA(+)) predominantly expressed functional neurotransmitter receptors for cholinergic and purinergic agonists while differentiating cells (TuJ1(+) and GFAP(+)) in the deeper layer (layer II) contained differentiated neurons and astrocytes and mainly responded to GABAergic and glutamatergic agonists and to veratridine, which activates voltage-dependent Na(+) channels. An active synaptic vesicle recycling was demonstrated by neuronal networks in the deeper layer using the endocytotic marker FM1-43. Cell polarization forming the characteristic two-layered structure was found to associate with the bFGF and FGF receptor signaling. These engineered functional tissue constructs have a potential use as tissue surrogates for drug screening and detection of environmental toxins, and in neural cell replacement therapy.

Members of the NuRD chromatin remodeling complex interact with AUF1 in developing cortical neurons.

Chromatin remodeling plays an important role in coordinating gene expression during cortical development, however the identity of molecular complexes present in differentiating cortical neurons that mediate the process remains poorly understood. The A + U-rich element-binding factor 1 (AUF1) is a known regulator of messenger RNA stability and also acts as a transcription factor upon binding to AT-rich DNA elements. Here we show that AUF1 is specifically expressed in subsets of proliferating neural precursors and differentiating postmitotic neurons of the developing cerebral cortex. Moreover, AUF1 is coexpressed with histone deacetylase 1 (HDAC1) and metastasis-associated protein 2 (MTA2), members of the nucleosome remodeling and histone deacetylase complex. AUF1 specifically and simultaneously binds to HDAC1, MTA2, and AT-rich DNA element, its gene regulatory function is modulated by the extent of histone acetylation and in animals lacking AUF1, the composition of the complex is modified. These results suggest that AUF1 is involved in integrating genetic and epigenetic signals during cortical development through recruiting HDAC1 and MTA2 to AT-rich DNA elements.

Self-renewing and differentiating properties of cortical neural stem cells are selectively regulated by basic fibroblast growth factor (FGF) signaling via specific FGF receptors.

Developmental processes mediating the initiation of lineage commitment from self-renewing neural stem cells (NSCs) remain mostly unclear because of the persisting ambiguity in identifying true NSCs from proliferative lineage-restricted progenitors (LRPs), which are directly or indirectly derived from NSCs. Our multilineage immunohistochemical analyses of early embryonic rat telencephalon at the onset of neurogenesis revealed clear dorsoventral gradients in the emergence of two types of neuronal progenitors (NPs) from multilineage-negative NSCs. Enumeration of NSCs using comprehensive flow cytometric analysis demonstrated that their precipitous decline in vivo involved both active differentiation into NPs and an increased propensity toward apoptosis. Both processes paralleled the dorsoventral changes in fibroblast growth factor receptor (FGFR) expressions. NSCs residing in the dorsal telencephalon coexpressed FGFR1 and FGFR3, whereas those residing in the ventral telencephalon also expressed FGFR2. NSCs exposed to basic fibroblast growth factor (bFGF) in vitro generated four stereotypical clonal expansion states: efficiently self-renewing, inefficiently self-renewing limited by apoptosis, exclusively neurogenic, and multipotential, generating up to five types of LRPs. The plasticity among these expansion states depended on ambient [bFGF], telencephalic developmental stage, and differential activation/inactivation of specific FGFRs. Coactivation of FGFR1 and FGFR3 promoted symmetrical divisions of NSCs (self-renewal), whereas inactivation of either triggered asymmetrical divisions and neurogenesis from these cells. Developmental upregulation of FGFR2 expression correlated with a shift of NSCs into a multipotential state or apoptosis. These results provide new insights regarding the roles of FGFRs in diversification of NSC properties and initiation of neural lineage-restricted differentiation.

Identification of a novel oligodendrocyte cell adhesion protein using gene expression profiling.

Oligodendrocytes undergo extensive changes as they differentiate from progenitors into myelinating cells. To better understand the molecular mechanisms underlying this transformation, we performed a comparative analysis using gene expression profiling of A2B5+ oligodendrocyte progenitors and O4+ oligodendrocytes. Cells were sort-purified ex vivo from postnatal rat brain using flow cytometry. Using Affymetrix microarrays, 1707 transcripts were identified with a more than twofold increase in expression in O4+ oligodendrocytes. Many genes required for oligodendrocyte differentiation were upregulated in O4+ oligodendrocytes, including numerous genes encoding myelin proteins. Transcriptional changes included genes required for cell adhesion, actin cytoskeleton regulation, and fatty acid and cholesterol biosynthesis. At the O4+ stage, there was an increase in expression of a novel proline-rich transmembrane protein (Prmp). Localized to the plasma membrane, Prmp displays adhesive properties that may be important for linking the extracellular matrix to the actin cytoskeleton. Together, our results highlight the usefulness of this discovery-driven experimental strategy to identify genes relevant to oligodendrocyte differentiation and myelination.

Canonical transient receptor potential 1 plays a role in basic fibroblast growth factor (bFGF)/FGF receptor-1-induced Ca2+ entry and embryonic rat neural stem cell proliferation.

Basic fibroblast growth factor (bFGF) and its major receptor FGF receptor-1 (FGFR-1) play an important role in the development of the cortex. The mechanisms underlying the mitogenic role of bFGF/FGFR-1 signaling have not been elucidated. Intracellular Ca2+ concentrations ([Ca2+]i) in proliferating cortical neuroepithelial cells are markedly dependent on Ca2+ entry (Maric et al., 2000a). The absence of voltage-dependent Ca2+ entry channels, which emerge later, indicates that other membrane mechanisms regulate [Ca2+]i during proliferation. Canonical transient receptor potential (TRPC) family channels are candidates because they are voltage independent and are expressed during CNS development (Strübing et al., 2003). Here, we investigated the involvement of TRPC1 in bFGF-mediated Ca2+ entry and proliferation of embryonic rat neural stem cells (NSCs). Both TRPC1 and FGFR-1 are expressed in the embryonic rat telencephalon and coimmunoprecipitate. Quantitative fluorescence-activated cell sorting analyses of phenotyped telencephalic dissociates show that approximately 80% of NSCs are TRPC1+, proliferating, and express FGFR-1. Like NSCs profiled ex vivo, NSC-derived progeny proliferating in vitro coexpress TRPC1 and FGFR1. Antisense knock-down of TRPC1 significantly decreases bFGF-mediated proliferation of NSC progeny, reduces the Ca2+ entry component of the Cai2+ response to bFGF without affecting Ca2+ release from intracellular stores or 1-oleoyl-2-acetyl-sn-glycerol-induced Ca2+ entry, and significantly blocks an inward cation current evoked by bFGF in proliferating NSCs. Both Ca2+ influx evoked by bFGF and NSC proliferation are attenuated by Gd3+ and SKF96365 two antagonists of agonist-stimulated Ca2+ entry. Together, these results show that TRPC1 contributes to bFGF/FGFR-1-induced Ca2+ influx, which is involved in self-renewal of embryonic rat NSCs.

Development of an ischemic tolerance model in a PC12 cell line.

Although ischemic tolerance has been described in a variety of primary cell culture systems, no similar in vitro models have been reported with any cell line. A model of ischemic preconditioning in the rat pheochromocytoma PC12 cell line is described here. When compared to nonpreconditioned cells, preexposure of PC12 cells to 6 hours of oxygen and glucose deprivation (OGD) significantly increased cell viability after 15 hours of OGD 24 hours later. Flow cytometry analysis of cells labeled with specific markers for apoptosis, Annexin V, and Hoechst 33342, and of DNA content, revealed that apoptosis is involved in OGD-induced PC12 cell death and that preconditioning of the cells mainly counteracts the effect of apoptosis. Immunocytochemistry of caspase-3, a central executioner in the apoptotic process, further confirmed the activation of apoptotic pathways in OGD-induced PC12 cell death. This model may be useful to investigate the cellular mechanisms involved in neuronal transient tolerance following ischemia.

Fluorescence-based sorting of neural stem cells and progenitors.

Neural stem cells (NSCs) are defined as undifferentiated cells originating from the neuroectoderm that have the capacity both to perpetually self-renew without differentiating and to generate multiple types of lineage-restricted progenitors (LRPs). LRPs can themselves undergo limited self-renewal and ultimately differentiate into highly specialized cells that make up the nervous system. However, this physiologically delimited definition of NSCs and LRPs has become increasingly blurred due to lack of protocols for effectively separating these types of cells from primary tissues. This unit discusses recent attempts using fluorescence-activated cell sorting (FACS) strategies to prospectively isolate NSCs from different types of LRPs as they appear in vivo, and details a protocol that optimally attains this goal. Thus, the strategy presented here provides a framework for more precise studies of NSC and LRP cell biology in the future, which can be applied to all vertebrates, including humans.

Neural stem cells redefined: a FACS perspective.

Using the generally accepted ontogenetic definition, neural stem cells (NSCs) are characterized as undifferentiated cells originating from the neuroectoderm that have the capacity both to perpetually self-renew without differentiating and to generate multiple types of lineage-restricted progenitors (LRP). LRPs can themselves undergo limited self-renewal, then ultimately differentiate into highly specialized cells that compose the nervous system. However, this physiologically delimited definition of NSCs has been increasingly blurred in the current state of the field, as the great majority of studies have retrospectively inferred the existence of NSCs based on their deferred functional capability rather than prospectively identifying the actual cells that created the outcome. Further complicating the matter is the use of a wide variety of neuroepithelial or neurosphere preparations as a source of putative NSCs, without due consideration that these preparations are themselves composed of heterogeneous populations of both NSCs and LRPs. This article focuses on recent attempts using FACS strategies to prospectively isolate NSCs from different types of LRPs as they appear in vivo and reveals the contrasting differences among these populations at molecular, phenotypic, and functional levels. Thus, the strategies presented here provide a framework for more precise studies of NSC and LRP cell biology in the future.

Pixel-based criteria-oriented analysis of time-lapse Ca2+-fluorescence images.

Since its inception, the analysis of time-lapse video-images acquired during Ca2+ imaging experiments using fluorescence microscopy has been progressively optimized for achieving a high temporal resolution. In contrast, the spatial resolution of the acquired images is often compromised during analysis to varying degrees by the need to draw regions of interest (ROI). We developed a strategy to analyze images at the acquired spatial resolution-pixel-by-pixel, grouping all pixels based on criteria of interest (COI) in regard to their associated fluorescence values over time and visualizing the distributions of the pixel-groups detected in a pseudo-colored map. We applied this pixel-based COI-strategy to the analysis of relative intracellular free calcium levels (Ca(i)(2+)) in attached cultured embryonic hippocampal cells under baseline and experimental conditions designed to evaluate the contribution of extracellular Ca2+ (Ca(e)(2+)) to baseline Ca(i)(2+) levels. We discovered distinct groups of Ca(e)(2+)-dependent Ca(i)(2+) regulation patterns emergent during the earliest phases of hippocampal cell differentiation, which were not limited to inter-cell differences. Thus, pixel-based COI-analysis of time-lapse images can be used to disclose distinct patterns of Ca(e)(2+)-dependent Ca(i)(2+) levels and their corresponding subcellular distributions in developing hippocampal cells. Such a strategy should be useful in studying the emergence and distribution of Ca(i)(2+) signaling at subcellular levels of resolution using fluorescence microscopy.

A role for semaphorins and neuropilins in oligodendrocyte guidance.

Oligodendrocytes develop in defined CNS regions as progenitor cells, which migrate to their final destinations, encountering soluble and membrane-bound signals that influence their differentiation and potential to myelinate axonal projections. To identify the regulatory genes that may be involved in this process, microarray analysis of developing oligodendroglia was performed. Several neural guidance genes, including members of the neuropilin (NP) and semaphorin families were detected. These findings were verified and expanded upon using RT-PCR with RNA from fluorescent activated cell sorted A2B5+ oligodendrocyte progenitors and O4+ pro-oligodendrocytes isolated from in vitro and in vivo sources. RT-PCR, western and immunocytochemical analyses revealed that oligodendrocytes expressed NP1, several alternatively spliced isoforms of NP2, and a broad spectrum of both soluble (Class 3), membrane-spanning (Class 4-6), and membrane-tethered (Class 7) semaphorin ligands. Class 3 semaphorins, in a modified stripe assay, caused the collapse of oligodendrocyte progenitor growth cones, redirection of processes, and altered progenitor migration. Our data support a role for neuropilins and semaphorins in orchestrating the migration patterns of developing oligodendrocytes in the CNS.

Prospective cell sorting of embryonic rat neural stem cells and neuronal and glial progenitors reveals selective effects of basic fibroblast growth factor and epidermal growth factor on self-renewal and differentiation.

We directly isolated neural stem cells and lineage-restricted neuronal and glial progenitors from the embryonic rat telencephalon using a novel strategy of surface labeling and fluorescence-activated cell sorting. Neural stem cells, which did not express surface epitopes characteristic of differentiation or apoptosis, were sorted by negative selection. These cells predominantly expressed fibroblast growth factor receptor type 1 (FGFR-1), and a minority exhibited basic fibroblast growth factor (bFGF), whereas few expressed epidermal growth factor receptor (EGFR) or EGF. Clonal analyses revealed that these cells primarily self-renewed without differentiating in bFGF-containing medium, whereas few survived or expanded in EGF-containing medium. Culturing of neural stem cells in bFGF- and EGF-containing medium permitted both self-renewal and differentiation into neuronal, astroglial, and oligodendroglial phenotypes. In contrast, lineage-restricted progenitors were directly sorted by positive selection using a combination of surface epitopes identifying neuronal or glial phenotypes or both. These cells were also primarily FGFR-1(+), with few EGFR(+), and most expanded and progressed along their expected lineages in bFGF-containing medium but not in EGF-containing medium. Ca(2+) imaging of self-renewing neural stem cells cultured in bFGF-containing medium revealed that bFGF, but not EGF, induced cytosolic Ca(2+) (Ca(2+)c) responses in these cells, whereas in bFGF- and EGF-containing medium, both bFGF and EGF evoked Ca(2+)c signals only in differentiating progeny of these cells. The results demonstrate that bFGF, but not EGF, sustains a calcium-dependent self-renewal of neural stem cells and early expansion of lineage-restricted progenitors, whereas together the two growth factors permit the initial commitment of neural stem cells into neuronal and glial phenotypes.

Sex-related differences in neuronal cell survival and signaling in rats.

While substantial and compelling evidence exists for sex-related differences in gross and microscopic brain structure, our understanding of the cellular mechanisms that give rise to these sexual dimorphisms is in its infancy. To investigate possible underlying mechanisms for sex-related differences in neuronal development, we examined neuronal survival and transductional signaling in male and female rat primary cortical neuronal cultures. We found that following 14 days in culture, the total number of surviving neurons was significantly higher in female cultures derived from either cortical plate (cp) or ventricular zone (vz), the regions where differentiating (cp) and proliferating (vz) cells are located. In addition, sex-related differences in the levels of phospho-ERK1 and Akt were also observed. Female cortical cultures had significantly higher levels of ERK1 in both cp and vz and higher levels of Akt in cp. No sex-related differences in Bcl-2 were observed. These data suggest that dimorphisms in cell survival may underlie enhanced neuronal survival (or decreased apoptosis) in female brain. Further, the appearance of sex-related differences at cellular and signaling levels in cortical neuronal cultures demonstrates that the effects of gender are not limited to parts of the brain mediating reproduction.

Presynaptic quantal plasticity: Katz's original hypothesis revisited.

Changes in the amplitudes of signals conveyed at synaptic contacts between neurons underlie many brain functions and pathologies. Here we review the possible determinants of the amplitude and plasticity of the elementary postsynaptic signal, the miniature. In the absence of a definite understanding of the molecular mechanism releasing transmitters, we investigated a possible alternative interpretation. Classically, both the quantal theory and the vesicle theory predict that the amount of transmitter producing a miniature is determined presynaptically prior to release and that rapid changes in miniature amplitude reflect essentially postsynaptic alterations. However, recent data indicates that short-term and long-lasting changes in miniature amplitude are in large part due to changes in the amount of transmitter in individual released packets that show no evidence of preformation. Current representations of transmitter release derive from basic properties of neuromuscular transmission and endocrine secretion. Reexamination of overlooked properties of these two systems indicate that the amplitude of miniatures may depend as much, if not more, on the Ca(2+) signals in the presynaptic terminal than on the number of postsynaptic receptors available or on vesicle's contents. Rapid recycling of transmitter and its possible adsorption at plasma and vesicle lumenal membrane surfaces suggest that exocytosis may reflect membrane traffic rather than actual transmitter release. This led us to reconsider the disregarded hypothesis introduced by Fatt and Katz (1952; J Physiol 117:109-128) that the excitability of the release site may account for the "quantal effect" in fast synaptic transmission. In this case, changes in excitability of release sites would contribute to the presynaptic quantal plasticity that is often recorded.

Functional properties of ryanodine receptors in hippocampal neurons change during early differentiation in culture.

6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid ryanodine (BODIPY-ryanodine) binding and Ca(2+) imaging were used to study the properties of ryanodine receptors (RyRs) and cytoplasmic Ca(2+) (Ca) changes in neurons cultured from the embryonic rat hippocampus during the earliest stages of differentiation. Baseline Ca levels declined from 164 +/- 5 (SD) nM at early stages to 70 +/- 4 nM in differentiated neurons. Fluorescent BODIPY-ryanodine binding signals identified activated RyRs in somata, which were eliminated by removal of external Ca(2+) or by blockage of Ca(2+) entry through L-type but not N-type Ca(2+) channels. The GABA synthesis inhibitor 3-mercaptopropionic acid completely abolished ryanodine binding. Caffeine or K(+)-depolarization inhibited the activity of RyRs at very early stages of differentiation but had stimulatory effects at later stages after a network of processes had formed. BayK-8644 stimulated RyRs throughout all regions of all differentiating cells. The results suggest that in differentiating embryonic hippocampal neurons the activity of RyRs is maintained via Ca(2+) entering through L-type Ca(2+) channels. The mode of activation of L-type voltage-gated Ca(2+) channels with either membrane depolarization or specific pharmacological agents affects the coupled activity of RyRs differently as neurons differentiate processes and networks.

Allopregnanolone activates GABA(A) receptor/Cl(-) channels in a multiphasic manner in embryonic rat hippocampal neurons.

Although 3alpha-substituted metabolites of progesterone are well established to interact with GABA(A) receptor/Cl(-) channels, the nature of the interaction(s) remains uncertain. We used patch-clamp recording to study the interaction with GABA(A) receptor/Cl(-) channels expressed by embryonic hippocampal neurons differentiating in culture and nonneuronal cells transfected with GABA(A) receptor subunits. Allopregnanolone primarily induced multiphasic current responses in neurons, which were eliminated by bicuculline, an antagonist of GABA at GABA(A) receptor/Cl(-) channels. Similar multiphasic responses blocked by bicuculline were induced by allopregnanollone in nonneuronal cells transfected with alpha(1) and gamma(2) subunits, indicating that the steroid activation of GABA(A) receptor/Cl(-) channels occurred independently of GABA. Fluctuation analyses of current responses to allopregnanolone and GABA revealed underlying channel activities with similar estimated unitary properties. However, although both agonists activated Cl(-) channels with similar estimated short and long burst-length durations, most of those stimulated by the steroid were short, while most of those opened by GABA were long. Allopregnanolone potentiated GABA-evoked Cl(-) currents in nonneuronal cells transfected with alpha(1) and beta(2) or beta(3) subunits, which did not exhibit multiphasic responses to the steroid, indicating another, independent action of the steroid at activated receptors. Pertussis toxin treatment eliminated the low-amplitude current and attenuated the high-amplitude current induced by allopregnanolone in a reversible manner. Mastoparan, which activates G proteins directly, triggered a high-amplitude current after a delay, which was blocked by bicuculline. The results indicate that allopregnanolone interacts with GABA(A) receptor/Cl(-) channels expressed by embryonic hippocampal neurons in multiple ways, some of which are mediated by G proteins.

Toluene inhibits muscarinic receptor-mediated cytosolic Ca2+ responses in neural precursor cells.

Toluene is widely used as a component in industrial solvents and many toluene-containing products are abused via inhalation. While many studies have demonstrated its inhibitory effects on neuronal activity, the effects of toluene on receptor signaling in proliferating and differentiating neural precursor cells are presently unclear. Here, using digital video microscopy and Ca2+ imaging, we investigated the effects of acute exposure to toluene on the function of muscarinic acetylcholine receptors (mAChRs) expressed in neural precursor cells. The neural precursor cells were isolatedfrom embryonic day 13 (E13) rat cortex and expanded in serum-free medium containing basic fibroblast growth factor (bFGF). We found that the acetylcholine (ACh) analog carbachol (CCh) induced a dose-dependent increase in cytosolic Ca2+, which was blocked by the muscarinic receptor antagonist atropine in a reversible manner. Toluene was added to the perfusion medium and concentrations of toluene in the medium were determined by gas chromatographic analysis. Following imaging, the cells were fixed and processed for 5-bromo-2'-deoxyuridine (BrdU, cell proliferation marker) and beta-tubulin (TuJ1, neuronal marker) immunostaining. In the 5 day culture, most cells continued to divide (BrdU+), while afew cells differentiated into young neurons (TuJ1-). The CCh-induced Ca2+ elevations in proliferating (BrdU+TuJ1-) neural precursor cells were significantly reduced by acute exposure to 0.15 mM toluene and completely blocked by 10 mM toluene. Toluene's inhibition of muscarinic receptor-mediated Ca2+ signaling was rapid, reversible and dose-dependent with an IC50 value 0.5 mM. Since muscarinic receptors mediate cell proliferation and differentiation during neural precursor cell development, these results suggest that depression of muscarinic signaling may play a role in toluene's teratogenic effect on the developing nervous system.